Difference: PPNegativeStaining (1 vs. 21)

Revision 2118 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Changed:
<
<

Protocols - Negative Staining

>
>

Protocols - Negative Staining

 

Jump to Principles

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • Carbon Coated Grid or PlasticCoatedGrid.
  • Pipette
  • Filter paper

Procedure:

  • Glow Discharge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of your chosen stain on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times
Changed:
<
<

Protocols - Deep Stain

>
>

Protocols - Deep Stain

 

You will need (in addition to items listed above):

  • 10% Trehalose
  • 5% bacitracin

Procedure:

Similar to negative staining with the additions to the stain:
  • Take 20ul of your chosen stain and add 2ul of trehalose and 2ul of bacitracin

Similar procedure to negative stain except you blot 4x:

  • Add a fourth drop of stain, and wick it from the edge with the tweezers, leaving the filter paper until the drop does not grow anymore
  • At this point, shake the grid vigorously to dry it as fast as possible

What to look for:

  • This procedure produces regions in which a significant amount of stain remains on the grid. In these regions, the particles can be completely embedded in a slab of stain and have excellent structural preservation. Aim to look for regions that appear like rivers, and take pictures at the edge of these, where the stain is deep enough to cover the particles, but not too thick.

  • These samples can also be prepared over small holes, in which case the stain film spans the hole and gives the same appearance of cryo negative staining.

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 2018 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Protocols - Negative Staining

Jump to Principles

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • Carbon Coated Grid or PlasticCoatedGrid.
  • Pipette
  • Filter paper

Procedure:

  • Glow Discharge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
Changed:
<
<
  • Place a drop of UA aqueous on the grid
>
>
  • Place a drop of your chosen stain on the grid
 
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Protocols - Deep Stain

You will need (in addition to items listed above):

  • 10% Trehalose
  • 5% bacitracin

Procedure:

Changed:
<
<
  • Glow Discharge grids
>
>		 Similar to negative staining with the additions to the stain:
Deleted:
<
<
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
 
  • Take 20ul of your chosen stain and add 2ul of trehalose and 2ul of bacitracin
Changed:
<
<
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
>
>		 Similar procedure to negative stain except you blot 4x:
Deleted:
<
<
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of the stain solution on the grid
  • Blot with filter paper immediately
  • Repeat the drop of stain and blotting a total of three times
 
  • Add a fourth drop of stain, and wick it from the edge with the tweezers, leaving the filter paper until the drop does not grow anymore
  • At this point, shake the grid vigorously to dry it as fast as possible

What to look for:

  • This procedure produces regions in which a significant amount of stain remains on the grid. In these regions, the particles can be completely embedded in a slab of stain and have excellent structural preservation. Aim to look for regions that appear like rivers, and take pictures at the edge of these, where the stain is deep enough to cover the particles, but not too thick.

  • These samples can also be prepared over small holes, in which case the stain film spans the hole and gives the same appearance of cryo negative staining.

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 1918 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Protocols - Negative Staining

Jump to Principles

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • Carbon Coated Grid or PlasticCoatedGrid.
  • Pipette
  • Filter paper

Procedure:

  • Glow Discharge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Protocols - Deep Stain

Changed:
<
<

You will need:

>
>

You will need (in addition to items listed above):

Deleted:
<
<
  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
 
  • 10% Trehalose
  • 5% bacitracin
Changed:
<
<
  • Carbon Coated Grid.
>
>
Deleted:
<
<
  • Pipette
  • Filter paper
 

Procedure:

  • Glow Discharge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Take 20ul of your chosen stain and add 2ul of trehalose and 2ul of bacitracin
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of the stain solution on the grid
  • Blot with filter paper immediately
  • Repeat the drop of stain and blotting a total of three times
  • Add a fourth drop of stain, and wick it from the edge with the tweezers, leaving the filter paper until the drop does not grow anymore
  • At this point, shake the grid vigorously to dry it as fast as possible

What to look for:

  • This procedure produces regions in which a significant amount of stain remains on the grid. In these regions, the particles can be completely embedded in a slab of stain and have excellent structural preservation. Aim to look for regions that appear like rivers, and take pictures at the edge of these, where the stain is deep enough to cover the particles, but not too thick.

  • These samples can also be prepared over small holes, in which case the stain film spans the hole and gives the same appearance of cryo negative staining.

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 1818 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Protocols - Negative Staining

Jump to Principles

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • Carbon Coated Grid or PlasticCoatedGrid.
  • Pipette
  • Filter paper

Procedure:

  • Glow Discharge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times
Added:
>
>

Protocols - Deep Stain

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • 10% Trehalose
  • 5% bacitracin
  • Carbon Coated Grid.
  • Pipette
  • Filter paper

Procedure:

  • Glow Discharge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Take 20ul of your chosen stain and add 2ul of trehalose and 2ul of bacitracin
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of the stain solution on the grid
  • Blot with filter paper immediately
  • Repeat the drop of stain and blotting a total of three times
  • Add a fourth drop of stain, and wick it from the edge with the tweezers, leaving the filter paper until the drop does not grow anymore
  • At this point, shake the grid vigorously to dry it as fast as possible

What to look for:

  • This procedure produces regions in which a significant amount of stain remains on the grid. In these regions, the particles can be completely embedded in a slab of stain and have excellent structural preservation. Aim to look for regions that appear like rivers, and take pictures at the edge of these, where the stain is deep enough to cover the particles, but not too thick.

  • These samples can also be prepared over small holes, in which case the stain film spans the hole and gives the same appearance of cryo negative staining.
 
Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 1703 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Protocols - Negative Staining

Jump to Principles

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • Carbon Coated Grid or PlasticCoatedGrid.
  • Pipette
  • Filter paper

Procedure:

  • Glow Discharge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 1603 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Protocols - Negative Staining

Jump to Principles

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
  • Carbon Coated Grid or PlasticCoatedGrid.
  • Pipette
  • Filter paper

Procedure:

Changed:
<
<
>
>
  • Glow Discharge grids
 
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 1503 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Protocols - Negative Staining

Jump to Principles

You will need:

  • Gloves
  • Parafilm
  • Tweezers
Changed:
<
<
>
>
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
 

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 1403 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Protocols - Negative Staining

Jump to Principles

You will need:

  • Gloves
  • Parafilm
  • Tweezers
  • Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
Changed:
<
<
>
>
 
  • Pipette
  • Filter paper

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 1303 Sep 2009 - Main.KdDerr

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

Principles & Protocols

Protocols - Negative Staining

Changed:
<
<

*Jump to Principles

>
>

Jump to Principles

 

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 1203 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:
<
<

Principles

Why Negative stain?

>
>

Protocols - Negative Staining

*Jump to Principles

Deleted:
<
<		Your sample will be subjected to a rather harsh environment in the electron microscope. First, an ultra high vacuum is needed to allow the electrons to travel freely through the microscope's column, and second, bombardment of the sample with high energy electrons tends to cause chemical changes in the specimens (radiation damage). However, proteins need water molecules to preserve their structural integrity, therefore something has to be done to counter the drying effects of the vacuum. The oldest approach discovered to this end, is to replace the water molecules with a salt. Using salts in which the counterion is a heavy metal provides not only protection against structural collapse, but also atoms that scatter electrons strongly, rendering biological samples easy to see. The term "negative stain" arises from the fact that heavy metal salts make the sample appear darker than the background.

What should I try?

There are many different compounds that can be tried as negative stains. The most common stain used for electron microscopy is uranyl acetate, since the uranium atoms produce a very strong contrast of the protein against the background. Other compounds commonmy tried are phosphotunstic acid (PTA), uranyl formate, ammonium molybdate, lead citrate, lead acetate, as well as commercial stains, such as Nano-Van and Nano-W (vanadate and tungsten salts respectively).

Conditions

Concentration, and pH are just two of the parameters that can be varied with negative stains. Uranyl salts (acetate and formate) tend to be quite acidic, and precipitate if the pH is brought closer to neutrality. At lower concentrations they tend to produce a smaller grain, revealing finer details, but making the preparation of good samples somewhat unreliable. PTA can be used at neutral pH, but reacts with phosphate buffers, producing images of poor quality. Nano-Van and Nano-W are stains that produce a rather fine grain, and are sold at neutral pH, however, not all samples stain well with these. The bottom line is that several stains should be tried with a given sample, and the best one chosen from direct evidence.

Is there more than one way to prepare negatively stained samples?

Unfortunately there are many different ways to prepare the samples, these being stained or ice embedded. Negatively stained samples are usually prepared on continuous carbon film (which itself can be prepared in several ways), but there are other methods that might be more suitable for other samples. Double carbon sandwiches can be made, deep stained samples, stain films over holes, etc. Factors to take into account include the type of data that needs to be collected (i.e., images for a publications, showing the size and shape of the molecule, or faithful projection images for a 3-D reconstruction).

Where do I get the negative stains to start?

At the NYSBC we are making an effort to keep a decent supply of stains to be used by our affiliates. We have stocks of uranyl acetate, uranyl formate (this has to be prepared fresh before using), ammonium molybdate, lead citrate, Nano-Van and Nano-W. Please remember that heavy metal salts tend to be highly toxic and a lot of caution should be exercised when using them (unless you want to become a heavy atom derivative). Please let us know if there is a stain you would like to try.

Protocols - Negative staining

 

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 1126 Aug 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtocStaining"
Deleted:
<
<		 NYSBC|Cryo-EM P&P: Staining - Negative 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:
>
>

Principles

Why Negative stain?

 
Changed:
<
<		 Staining
>
>		Your sample will be subjected to a rather harsh environment in the electron microscope. First, an ultra high vacuum is needed to allow the electrons to travel freely through the microscope's column, and second, bombardment of the sample with high energy electrons tends to cause chemical changes in the specimens (radiation damage). However, proteins need water molecules to preserve their structural integrity, therefore something has to be done to counter the drying effects of the vacuum. The oldest approach discovered to this end, is to replace the water molecules with a salt. Using salts in which the counterion is a heavy metal provides not only protection against structural collapse, but also atoms that scatter electrons strongly, rendering biological samples easy to see. The term "negative stain" arises from the fact that heavy metal salts make the sample appear darker than the background.
Deleted:
<
<

Protocols - Negative staining

 
Changed:
<
<		Contents
>
>

What should I try?

Added:
>
>		There are many different compounds that can be tried as negative stains. The most common stain used for electron microscopy is uranyl acetate, since the uranium atoms produce a very strong contrast of the protein against the background. Other compounds commonmy tried are phosphotunstic acid (PTA), uranyl formate, ammonium molybdate, lead citrate, lead acetate, as well as commercial stains, such as Nano-Van and Nano-W (vanadate and tungsten salts respectively).

Conditions

Concentration, and pH are just two of the parameters that can be varied with negative stains. Uranyl salts (acetate and formate) tend to be quite acidic, and precipitate if the pH is brought closer to neutrality. At lower concentrations they tend to produce a smaller grain, revealing finer details, but making the preparation of good samples somewhat unreliable. PTA can be used at neutral pH, but reacts with phosphate buffers, producing images of poor quality. Nano-Van and Nano-W are stains that produce a rather fine grain, and are sold at neutral pH, however, not all samples stain well with these. The bottom line is that several stains should be tried with a given sample, and the best one chosen from direct evidence.

Is there more than one way to prepare negatively stained samples?

Unfortunately there are many different ways to prepare the samples, these being stained or ice embedded. Negatively stained samples are usually prepared on continuous carbon film (which itself can be prepared in several ways), but there are other methods that might be more suitable for other samples. Double carbon sandwiches can be made, deep stained samples, stain films over holes, etc. Factors to take into account include the type of data that needs to be collected (i.e., images for a publications, showing the size and shape of the molecule, or faithful projection images for a 3-D reconstruction).

Where do I get the negative stains to start?

At the NYSBC we are making an effort to keep a decent supply of stains to be used by our affiliates. We have stocks of uranyl acetate, uranyl formate (this has to be prepared fresh before using), ammonium molybdate, lead citrate, Nano-Van and Nano-W. Please remember that heavy metal salts tend to be highly toxic and a lot of caution should be exercised when using them (unless you want to become a heavy atom derivative). Please let us know if there is a stain you would like to try.

Protocols - Negative staining

 

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 1012 Feb 2009 - Main.KakoliMitra

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

Contents

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 912 Feb 2009 - Main.KakoliMitra

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

Contents

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 812 Feb 2009 - Main.KakoliMitra

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

Contents

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"

Revision 712 Feb 2009 - Main.KakoliMitra

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

Contents

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
Added:
>
>
META TOPICMOVED by="KakoliMitra" date="1234476055" from="Main.NegativeStaining" to="Main.PPNegativeStaining"
 

Revision 612 Feb 2009 - Main.KakoliMitra

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

Contents

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

Public website mirror

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"

Revision 512 Feb 2009 - Main.KakoliMitra

 
META TOPICPARENT name="SamplePreparation"
Changed:
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<		Negaive Staining
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>
Added:
>
>		 NYSBC|Cryo-EM P&P: Staining - Negative staining
 
Changed:
<
<
>
>
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
 
Added:
>
>		 Staining

Protocols - Negative staining

Contents

 

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • Most negative stains are very toxic, especially Uranium salts.
  • Secure the grid in a pair of tweezers
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times
Added:
>
>
Public website mirror
 
Changed:
<
<
>
>
 
Deleted:
<
<		Visit Cryo EM Web site at http://www.nysbc.org/facilities/CEM
 -- DavidStokes - 18 Mar 2005
Deleted:
<
<
 
META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"

Revision 417 May 2007 - Main.RubenDiaz

 
META TOPICPARENT name="SamplePreparation"
Negaive Staining

You will need:

  • Gloves
  • Parafilm
  • Tweezers
Changed:
<
<
>
>
 
  • Pipette
  • Filter paper

Procedure:

Changed:
<
<
    • UA is very toxic
>
>
    • Most negative stains are very toxic, especially Uranium salts.
Deleted:
<
<
  • Pipette sample onto plastic coated grid
 
  • Secure the grid in a pair of tweezers
Added:
>
>
  • Pipette sample (~3 microliters) onto glow-discharged grid.
  • After about 30 seconds, blot the excess sample from the grid with filter paper
 
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

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

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"

Revision 309 Jun 2006 - Main.KdDerr

 
META TOPICPARENT name="SamplePreparation"
Changed:
<
<		Negaive Staining

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • UA is very toxic
  • Pipette sample onto plastic coated grid
  • Secure the grid in a pair of tweezers
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

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

-- DavidStokes - 18 Mar 2005

>
>		Negaive Staining

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • UA is very toxic
  • Pipette sample onto plastic coated grid
  • Secure the grid in a pair of tweezers
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

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

-- DavidStokes - 18 Mar 2005

 
META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"

Revision 222 Mar 2005 - Main.DavidStokes

 
META TOPICPARENT name="SamplePreparation"
Negaive Staining

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • UA is very toxic
  • Pipette sample onto plastic coated grid
  • Secure the grid in a pair of tweezers
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

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

-- DavidStokes - 18 Mar 2005

Added:
>
>
 
META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"

Revision 118 Mar 2005 - Main.DavidStokes

 
META TOPICPARENT name="SamplePreparation"
Negaive Staining

You will need:

Procedure:

  • GlowDischarge grids
  • Put on gloves!
    • UA is very toxic
  • Pipette sample onto plastic coated grid
  • Secure the grid in a pair of tweezers
  • Place a drop of UA aqueous on the grid
  • Blot with filter paper immediately
  • Repeat the drop of UA aq and blotting a total of three times

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

-- DavidStokes - 18 Mar 2005

META FILEATTACHMENT attr="" comment="" date="1111178486" name="UAstain.doc" path="UA stain.doc" size="19968" user="DavidStokes" version="1.1"
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