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Cryostat sectioning

Standard Protocols

Cryostat Sectioning

  1. Make sure the temperature of the main cryostat chamber is at –150C and that the quick freeze compartment is at least -400C.
  2. Always carry your tissue samples across in liquid nitrogen
  3. Remove the blade from the freezer next to the cryostat and slide it into its holder in the cryostat. The writing on the back of the blade should be facing up. Adjust the angle of the blade to 150 and tighten the screws to hold the blade in place.
  4. Remove the plastic lid from the quick-freeze compartment. Underneath should be the stage and some razor blades.
  5. Place your tissue sample in the quick-freeze compartment and fracture it into smaller pieces uses the razor blades. The size of tissue to be sectioned should be no larger than 1 cm3. The smaller the tissue the better the quality of the section, especially when making thin sections.
  6. Place some Cryo-M-Bed embedding compound onto the stage (enough to support the tissue sample). Place the sample into the embedding compound then cover with Cryo-M-Bed. Freeze the sample with the Cryospray.
  7. Place the stage on the stage clamp above the blade. Move the stage backwards with the wheel so that the sample is behind the blade.
  8. Set the thickness of the sections to 20 microns and turn the large wheel on the right hand side of the cryostat anti-clockwise. Once the blade starts to cut into the section adjust the thickness to whatever is appropriate.
  9. Place the roll-bar onto the blade and turn the handle in a smooth, steady motion.
  10. Once a section has been cut, lift the roll bar to expose the scetion and gently place a slide on top of it. The section should then stick to the slide.
  11. Leave the slides to air-dry before use. Slides can be stored at -200 C until required.

 

 

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