Difference: PPPositiveStaining (1 vs. 25)

Revision 2511 Mar 2010 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Positive Staining

Jump to Principles

You will need:

  • Gloves
  • Tweezers
  • Timer
  • DD H2O
  • Beakers
  • Uranyl Acetate
  • Lead Stain
  • Pipette
  • Sample grids

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast
Changed:
<
<

Comparison between stains

>
>

Comparison between EtOH? and Aq stains

 

Uranyl Acetate in EtOH?

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain
Changed:
<
<

Lead Citrate (preferred)

>
>
Added:
>
>

Lead Citrate Recipe

 
  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
  • Stain samples for 30 sec to 15 minutes
  • Cover sample with Petri dish when staining to prevent precipitation
Public website mirror

-- DavidStokes - 18 Mar 2005

META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 2418 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Positive Staining

Jump to Principles

You will need:

  • Gloves
  • Tweezers
  • Timer
  • DD H2O
  • Beakers
  • Uranyl Acetate
  • Lead Stain
  • Pipette
  • Sample grids

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH?

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
  • Stain samples for 30 sec to 15 minutes
  • Cover sample with Petri dish when staining to prevent precipitation
Public website mirror

-- DavidStokes - 18 Mar 2005

META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 2318 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Positive Staining

Jump to Principles

You will need:

  • Gloves
  • Tweezers
  • Timer
  • DD H2O
  • Beakers
  • Uranyl Acetate
  • Lead Stain
  • Pipette
  • Sample grids

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH?

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
  • Stain samples for 30 sec to 15 minutes
Changed:
<
<
  • Cover sample with Petri dish when staining to prevent percipitation
>
>
  • Cover sample with Petri dish when staining to prevent precipitation
 
Public website mirror

-- DavidStokes - 18 Mar 2005

META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 2218 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Positive Staining

Jump to Principles

You will need:

  • Gloves
  • Tweezers
  • Timer
  • DD H2O
  • Beakers
  • Uranyl Acetate
  • Lead Stain
  • Pipette
  • Sample grids

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Changed:
<
<

Uranyl Acetate in EtOH

>
>

Uranyl Acetate in EtOH?

 
  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.
Changed:
<
<

Aqueous Uranyl Acetate

>
>

Aqueous Uranyl Acetate

 
  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
  • Stain samples for 30 sec to 15 minutes
  • Cover sample with Petri dish when staining to prevent percipitation
Public website mirror

-- DavidStokes - 18 Mar 2005

META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 2118 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Positive Staining

Jump to Principles

You will need:

  • Gloves
  • Tweezers
  • Timer
  • DD H2O
  • Beakers
  • Uranyl Acetate
  • Lead Stain
  • Pipette
  • Sample grids

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
  • Stain samples for 30 sec to 15 minutes
  • Cover sample with Petri dish when staining to prevent percipitation
Public website mirror

-- DavidStokes - 18 Mar 2005

Deleted:
<
<
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
 
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 2018 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Positive Staining

Jump to Principles

You will need:

  • Gloves
  • Tweezers
  • Timer
  • DD H2O
  • Beakers
  • Uranyl Acetate
  • Lead Stain
  • Pipette
  • Sample grids

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
  • Stain samples for 30 sec to 15 minutes
  • Cover sample with Petri dish when staining to prevent percipitation
Public website mirror

-- DavidStokes - 18 Mar 2005

Deleted:
<
<
META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
 
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 1918 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Positive Staining

Jump to Principles

You will need:

  • Gloves
  • Tweezers
  • Timer
  • DD H2O
  • Beakers
  • Uranyl Acetate
  • Lead Stain
  • Pipette
  • Sample grids

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
  • Stain samples for 30 sec to 15 minutes
  • Cover sample with Petri dish when staining to prevent percipitation
Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
Deleted:
<
<
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
 
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 1818 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Positive Staining

Jump to Principles

You will need:

  • Gloves
  • Tweezers
  • Timer
  • DD H2O
  • Beakers
  • Uranyl Acetate
  • Lead Stain
  • Pipette
  • Sample grids

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
  • Stain samples for 30 sec to 15 minutes
  • Cover sample with Petri dish when staining to prevent percipitation
Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
Deleted:
<
<
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
 
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 1716 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CryoSP"
Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Positive Staining

Jump to Principles

You will need:

  • Gloves
  • Tweezers
  • Timer
  • DD H2O
  • Beakers
  • Uranyl Acetate
Changed:
<
<
>
>
  • Lead Stain
 
  • Pipette
  • Sample grids

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain
Deleted:
<
<

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation
 

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
Added:
>
>
  • Stain samples for 30 sec to 15 minutes
  • Cover sample with Petri dish when staining to prevent percipitation
 
Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 1603 Sep 2009 - Main.KdDerr

Changed:
<
<
META TOPICPARENT name="CemProtocStaining"
>
>
META TOPICPARENT name="CryoSP"
 Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

Principles & Protocols

Protocols - Positive Staining

Jump to Principles

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 1503 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtocStaining"
Added:
>
>		Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM
 

Principles & Protocols

Changed:
<
<

Positive Staining

>
>

Protocols - Positive Staining

Jump to Principles

 

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 1421 Aug 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtocStaining"
Deleted:
<
<		 NYSBC|Cryo-EM P&P: Staining - Positive Staining
 
Changed:
<
<		Principles & Protocols
>
>

Principles & Protocols

 
Deleted:
<
<
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? CEM Introduction Microscope Schedule NYSBC Directory Cryo-EM public website
 
Added:
>
>

Positive Staining

 
Deleted:
<
<		 Staining

Protocols - Positive Staining

Contents

 

You will need:

  • Gloves
  • Tweezers
  • Timer
  • DD H2O
  • Beakers
Changed:
<
<
>
>
  • Uranyl Acetate
 

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
  • Filter each stain through a syringe filter before using
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 1319 Jun 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtocStaining"
NYSBC|Cryo-EM P&P: Staining - Positive Staining

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? CEM Introduction Microscope Schedule NYSBC Directory Cryo-EM public website

Staining

Protocols - Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
  • Place Wet filter paper in a petri dish
  • Parafilm on top
  • Al foil cover
Added:
>
>
  • Filter each stain through a syringe filter before using
 
  • 20 min 4% UA Stain (aqueous)
  • Rinse well in DD H2O
  • 3 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Lead Citrate (preferred)

  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 1219 Jun 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtocStaining"
NYSBC|Cryo-EM P&P: Staining - Positive Staining

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? CEM Introduction Microscope Schedule NYSBC Directory Cryo-EM public website

Staining

Protocols - Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
Changed:
<
<
  • 10 min UA Stain (aqueous)
>
>
  • Place Wet filter paper in a petri dish
Added:
>
>
  • Parafilm on top
  • Al foil cover
  • 20 min 4% UA Stain (aqueous)
 
  • Rinse well in DD H2O
Changed:
<
<
  • 5 min lead stain
>
>
  • 3 min lead stain
 
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation
Changed:
<
<
>
>

Lead Citrate (preferred)

Added:
>
>
  • 0.01g in 10mL D20
  • 3 drops of 10 N NaOH?
 
Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 1119 Jun 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtocStaining"
NYSBC|Cryo-EM P&P: Staining - Positive Staining

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? CEM Introduction Microscope Schedule NYSBC Directory Cryo-EM public website

Staining

Protocols - Positive Staining

Contents

You will need:

  • Gloves
Deleted:
<
<
  • Parafilm
 

Procedure:

  • Put on gloves! (stains are very toxic)
  • 10 min UA Stain (aqueous)
  • Rinse well in DD H2O
  • 5 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 1012 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocStaining"
NYSBC|Cryo-EM P&P: Staining - Positive Staining

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
Changed:
<
<
CEMfac Home NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
>
>
CEMfac Home? CEM Introduction Microscope Schedule NYSBC Directory Cryo-EM public website
 

Staining

Protocols - Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
  • 10 min UA Stain (aqueous)
  • Rinse well in DD H2O
  • 5 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 912 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocStaining"
NYSBC|Cryo-EM P&P: Staining - Positive Staining

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
Changed:
<
<
CEMfac Home NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
>
>
CEMfac Home NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
 

Staining

Protocols - Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
  • 10 min UA Stain (aqueous)
  • Rinse well in DD H2O
  • 5 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 812 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocStaining"
NYSBC|Cryo-EM P&P: Staining - Positive Staining

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
Changed:
<
<
CEMfac Home? NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
>
>
CEMfac Home NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
 

Staining

Protocols - Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
  • 10 min UA Stain (aqueous)
  • Rinse well in DD H2O
  • 5 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"

Revision 712 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="CemProtocStaining"
NYSBC|Cryo-EM P&P: Staining - Positive Staining

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website

Staining

Protocols - Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
  • 10 min UA Stain (aqueous)
  • Rinse well in DD H2O
  • 5 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
Added:
>
>
META TOPICMOVED by="KakoliMitra" date="1234475548" from="Main.PositiveStaining" to="Main.PPPositiveStaining"
 

Revision 612 Feb 2009 - Main.KakoliMitra

Changed:
<
<
META TOPICPARENT name="SamplePreparation"
>
>
META TOPICPARENT name="CemProtocStaining"
  NYSBC|Cryo-EM P&P: Staining - Positive Staining

Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website

Staining

Protocols - Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
  • 10 min UA Stain (aqueous)
  • Rinse well in DD H2O
  • 5 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"

Revision 512 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="SamplePreparation"
Changed:
<
<		Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM
>
>
Added:
>
>		 NYSBC|Cryo-EM P&P: Staining - Positive Staining
 
Changed:
<
<		Positive Staining
>
>
Added:
>
>		Principles & Protocols
Interested in EM? Using CEMfac? Principles & Protocols CEMfac Equipment Seminars & Courses
CEMfac Home? NYSBC & Cryo-EM Information? Microscope Schedule NYSBC Directory Cryo-EM public website
 
Changed:
<
<		Contents
>
>		 Staining
Added:
>
>

Protocols - Positive Staining

 
Added:
>
>		Contents
 
Deleted:
<
<
 

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
  • 10 min UA Stain (aqueous)
  • Rinse well in DD H2O
  • 5 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation
Added:
>
>
Public website mirror
 
Added:
>
>
 
Deleted:
<
<

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

 -- DavidStokes - 18 Mar 2005
Deleted:
<
<
 
META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"

Revision 409 Jun 2006 - Main.KdDerr

 
META TOPICPARENT name="SamplePreparation"
Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

Positive Staining

Contents

You will need:

Added:
>
>
  • Gloves
 

Procedure:

  • Put on gloves! (stains are very toxic)
  • 10 min UA Stain (aqueous)
  • Rinse well in DD H2O
  • 5 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"

Revision 309 Jun 2006 - Main.KdDerr

 
META TOPICPARENT name="SamplePreparation"
Changed:
<
<		Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic
  • 1 min UA Stain
  • Rinse well in DD H2O
  • 1 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="Reynold Lead Citrate Stain" date="1111177174" name="ReynoldsLeadCitrateStain.doc" path="Reynolds Lead Citrate Stain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" comment="Sato stain" date="1111177197" name="Satostain.doc" path="Sato stain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" comment="Uranyl Acetate stain" date="1111177230" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" comment="UA and Reynolds stain" date="1111177258" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="Uranyl Acetate and Reynolds Lead Citrate Staining.doc" size="37376" user="DavidStokes" version="1.1"
>
>		Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic)
  • 10 min UA Stain (aqueous)
  • Rinse well in DD H2O
  • 5 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" autoattached="1" comment="Reynold Lead Citrate Stain" date="1144161306" name="ReynoldsLeadCitrateStain.doc" path="ReynoldsLeadCitrateStain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="Sato stain" date="1144161306" name="Satostain.doc" path="Satostain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" autoattached="1" comment="UA and Reynolds stain" date="1144161306" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="UranylAcetateandReynoldsLeadCitrateStaining.doc" size="37376" user="DavidStokes" version="1.1"
Added:
>
>
META FILEATTACHMENT attr="" autoattached="1" comment="Uranyl Acetate stain" date="1144161306" name="UAstain.doc" path="UAstain.doc" size="19968" user="DavidStokes" version="1.1"
 

Revision 222 Mar 2005 - Main.DavidStokes

 
META TOPICPARENT name="SamplePreparation"
Changed:
<
<		Cryo EM Web site at http://www.nysbc.org/facilities/CEM
>
>		Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM
 Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic
  • 1 min UA Stain
  • Rinse well in DD H2O
  • 1 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

Added:
>
>
 
META FILEATTACHMENT attr="" comment="Reynold Lead Citrate Stain" date="1111177174" name="ReynoldsLeadCitrateStain.doc" path="Reynolds Lead Citrate Stain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" comment="Sato stain" date="1111177197" name="Satostain.doc" path="Sato stain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" comment="Uranyl Acetate stain" date="1111177230" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" comment="UA and Reynolds stain" date="1111177258" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="Uranyl Acetate and Reynolds Lead Citrate Staining.doc" size="37376" user="DavidStokes" version="1.1"

Revision 118 Mar 2005 - Main.DavidStokes

 
META TOPICPARENT name="SamplePreparation"
Cryo EM Web site at http://www.nysbc.org/facilities/CEM

Positive Staining

Contents

You will need:

Procedure:

  • Put on gloves! (stains are very toxic
  • 1 min UA Stain
  • Rinse well in DD H2O
  • 1 min lead stain
  • Rinse well DD H2O

Tips

  • Longer times can be used if necessary.
  • Resist the urge to over stain it only results in artifacts
  • Learn to rely on the microscope to provide ample contrast

Comparison between stains

Uranyl Acetate in EtOH

  • Primarily used when samples difficult to stain (i.e. Spurr's resin)
  • Penetrates more easily stain for 1-15 minutes
  • Grids will sink to bottom of UA so place grid sample side up
  • Shelf life is not as long.
  • More risk of UA precipitating out of solution
  • Stain with UA in ETOH before lead to minimize staining artifacts.

Aqueous Uranyl Acetate

  • Penetrates more slowly stain for 1min-1 hour
  • Provides ample contrast when paired with lead stain

Reynolds Lead Citrate Stain

  • Stain samples for 30 sec to 15 minutes
  • Appears to be less stable in the fridge than SATO stain

Sato Stain

  • Stain samples for 30 sec to 15 minutes
  • More contamination is evident on grids stained with SATO
  • Cover sample with Petri dish when staining to prevent percipitation

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="Reynold Lead Citrate Stain" date="1111177174" name="ReynoldsLeadCitrateStain.doc" path="Reynolds Lead Citrate Stain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" comment="Sato stain" date="1111177197" name="Satostain.doc" path="Sato stain.doc" size="20480" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" comment="Uranyl Acetate stain" date="1111177230" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META FILEATTACHMENT attr="" comment="UA and Reynolds stain" date="1111177258" name="UranylAcetateandReynoldsLeadCitrateStaining.doc" path="Uranyl Acetate and Reynolds Lead Citrate Staining.doc" size="37376" user="DavidStokes" version="1.1"
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