Difference: CemProtocHPFU (24 vs. 25)

Revision 2503 Sep 2009 - Main.KdDerr

 
META TOPICPARENT name="CemProtoc"
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Principles & Protocols

Principles - High Pressure Freezing

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Biological specimens thicker than 250 nm will not form vitreous ice without the use of cryo protectants under normal atmospheric conditions. A high pressure freezer is used to raise the pressure on a sample allowing it to be frozen with a jet of liquid nitrogen. Liquid nitrogen cannot normally be used for freezing because of the Leidenfrost effect; the instantaneous boiling of the liquid nitrogen creating an insulating vapor layer when it comes in contact with the warmer sample. Increasing the pressure raises the vitreous freezing temperature. After freezing, the sample can either be directly sectioned (cryo-ultramicrotomy) for visualization in the frozen hydrated state, or subjected to freeze substitution and resin infiltration followed by conventional ultramicrotomy.

Filler selection - Looking for an osmotically inactive hydrophilic substance

Penetrating Fillers

  • Glycerol
  • Ethylene glycol

Non Penetrating Fillers

  • 1-Hexadecene
  • BSA (10-20%)
  • Polyvinylpyrrolidone (PVP) (15%)
  • Dextran (15-25%)
  • Ficoll (5-15%)
  • low-melt agarose (0.5-2%)
  • Cold water fish gelatin (50-100%)
  • Yeast comes from Drosophila food.
  • Bakers yeast - Rehydrate with buffer or culture media and stir with a toothpick until like thin peanut butter

Loading

  • Use 1-2% agar as work surface. Prevents drying
  • Lower hat with cells facing up
  • Spacer grids
    • Slot grid or "Chein" grids (EMS or Pella):
    • 27 um thickness or
    • rhodium maxteform spacer, silver 50 um thickness
  • hat on top is preincubated with hexadecene - set it on filter paper saturated with hexadecene in Petri dish.
  • Max dimension for specimen between hats is 500-600 um
  • Use eyeliner brush to remove large amounts of fluid; paper strips for small amounts
  • Hats come apart easily under LN2. Sample stays with slot if a suspension, or on surface of bottom hat if a culture. Or on bottom hat if top has been treated with hexadecene. Hexadecene seems to be a good lubricant to keep hats from sticking to the holder.

Embedding

  • en face.
    • Could use flat molds and place hats at the tip and try to cut in cross section.
    • reembedding. For en face sections, embed hats or sapphire disks in tip of Beem capsule.
  • sapphire disk tricks
    • Trim away resin such that it is not covering sides of hat or sapphire at all. Even undercut as much as possible.
    • Dip in LN2 or even LN2 vapors. Some people dip multiple times (cycles of freeze thaw).
    • Grab disk face on with hemostats or fine pliers and pull apart.

Substitution

  • Standard mixture: 1% OsO4, 0.1% glutaraldehyde in acetone
    • 72hrs at -90degC followed by overnight at either -60 or -30 deg C
    • temp gradients at 5deg/hr
  • Other variations:
    • 5 hr at -90 was ok for recent studies of fly eggs
    • -90 for 8 hrs, -60 for 8 hrs, -30 for 8 hrs

  • 3 washes with acetone, ~10 min each

Protocol for yeast

  • Log-phase growth (OD 0.3-0.5)
  • Filter under suction vacuum on 25 mm polycarbonate (does it matter) filter 0.4 um
  • Place filter on wet filter paper or agar
  • Make spatula out of toothpick by cutting across the tip
  • Scrape up a bit of yeast from the filter and place in the hat (100 um well)
  • Cover with hexadecene-coated flat
  • Yeast stays in the 100um well after freezing
  • During freeze substitution, yeast floats away from the hat, visible as thin brown disk in the acetone solution.

Protocols

  • Set ALLOWTOPICVIEW =

-- BillRice - 10 Aug 2009

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