Cryostat Notes
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Gabriel Kaufman 29 July 2011 Fritz Lab Cryostat notes Preparation Equilibrate specimen blocks in cryostat or in -20°C freezer for at least 1 hour prior to sectioning them. Prepare around 300 mL of 100% acetone in glass staining dish in refrigerator (use chemical-resistant tray). Specimen handling Mount specimen on chuck using OCT. Insert chuck into object holder and tighten retaining screw. Check temperatures and adjust as necessary: chamber temperature (CT) should range from −25°C to −20°C and object - 1 -
Transcript of Cryostat Notes
Gabriel Kaufman 29 July 2011Fritz Lab
Cryostat notes
Preparation
Equilibrate specimen blocks in cryostat or in -20°C freezer for at least 1 hour prior to sectioning them.
Prepare around 300 mL of 100% acetone in glass staining dish in refrigerator (use chemical-resistant tray).
Specimen handling
Mount specimen on chuck using OCT.
Insert chuck into object holder and tighten retaining screw.
Check temperatures and adjust as necessary: chamber temperature (CT) should range from −25°C to −20°C and object temperature (OT) should range from −18°C to −15°C, depending on tissue being sectioned.
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Gabriel Kaufman 29 July 2011Fritz Lab
Orient specimen in diamond fashion, so that the smallest area of specimen hits the blade first. DO NOT adjust clearance angle or specimen x-y orientation – trim instead.
Sectioning
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Gabriel Kaufman 29 July 2011Fritz Lab
Trim at 30 µm until the entire tissue is exposed.
Section at 5-10 µm, depending upon the tissue type. Mount sections on SuperFrost Plus slides. One can mount multiple sections on the same slide (e.g. serial sections from the same block, control and experimental blocks, etc.).
Block handling
When finished sectioning, remove block from chuck using a razor blade. Re-wrap block in foil and store at −80°C.
Slide handling
MAKE SURE to label slides in pencil. Air-dry slides in glass rack after mounting sections on them.
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Gabriel Kaufman 29 July 2011Fritz Lab
When finished sectioning for the day, and all slides have been air-dried for at least 15 minutes, fix the slides in cold acetone (in the refrigerator) for precisely 7 minutes (use a timer!!).
This fixation step must always be performed, even when sectioning tissues from a fixative-perfused animal.
Remove slides from acetone, and let them dry for at least a half-hour (can be left overnight).
[Recover acetone into a glass bottle using a glass funnel – acetone will dissolve plastic.]
Rack dry fixed slides in a plastic slide box. Store the box in a plastic zip-lock bag with a little dessicant (Drierite) at −80°C.
Stain slides as soon as reasonably possible: fresh slides stain better.
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