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CryosectioningEmbryos.md

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CryosectioningEmbryos.md

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Cryosectioning Embryos

Reagents

*	Gasket stencil
*	Delicate Task Wiper (The big sheet of Kimwipe)
*	Ethanol
*	Chromium Gelatin
*	Slide Holder Box
*	Paintbrush
* 	OCT Compound
*	Blade - Cryosectioning Blade and Standard Blade
*	Slides - Circle Coverslip Slides and Standard Microscope Slides

Preparing Slides for Cryosectioning

  1. On a piece of napkin, make a stencil of the gasket for the coverslip slides. Dots around the circle, center, two lines at the poles.
  2. Place a Delicate Task Wiper on top of working station.
  3. Take a circle coverslip and wipe both sides down with ethanol, leave to dry
  4. Mark two notches on both poles of the slide
  5. Pipette 500uL of Chromium Gelatin onto slide in a line over the center. Leave for 30-45 minutes
  6. Aspirate
  7. Place in Slide Holder Box. Keep track of what side is the front and back
  8. Store at room temperature

Setting Up The Cryosectioning Machine

  1. Make sure the cyrosection station is at the "home" position. (Light should be blinking). If not press the home position.
  2. Do not press the defrost button!!!!
  3. Place the cryosectioning blade inside the chamber - needs to be cold be the time sectioning starts
  4. Place your paintbrush inside the chamber
  5. Take out the circular stage and warm it up with your hands.
  6. Once warm, squeeze OCT (in a swirl motion) and leave it in a flat surface
  7. Pop off the frozen embryo from the cryomold and quickly mark the notches with a marker. Place it onto to OCT compound and make sure the embryos side is closer to you and not touching the circle stage
  8. Place the circle stage back into the chamber, clamp it down and wait for the OCT on the circle stage to freeze. Can remove the clamp after the OCT starts freezing
  9. Once frozen, lock the stage onto the block holder and lock it in place
  10. With the standard blade, carve out the frozen OCT into a rectangle using the notches as a mark
  11. Make one more carve at the bottom of the rectangular frozen OCT to make it a V-shape. This will help with the paintbrush step
  12. Place the cryosectioning blade into it's locking position

Cutting Through The Embryo

  1. Set the machine to cut 6 microns
  2. Switch the safety lock and turn the handle to make the frozen OCT come in close contact with the blade. Adjust the knobs for proper slice angle
  3. Once at the proper position, make the first cut into your embryo by turning the handle away from you while pulling the section down with the paintbrush. Don't cut all the way! Turn the handle towards you to raise the section. You can flatten it a bit with the paintbrush
  4. Transfer the section sample onto a standard microscope slide to check if you've reached your embryo. Once you have start transfering sections onto circular coverslip slides
  5. Keep cutting away through the whole sample

Clean-up

  1. Collect all frozen OCT sections onto the metal tray and throw away in trash
  2. Remove the circle stage from the block holder and leave it in room temp to let the OCT thaw a bit. Once it's soft, take the blade and remove the frozen OCT. Put in trash
  3. Set the machine back to its home setting
  4. Clean up everything inside the machine for the next user