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EmbeddingEmbryos.md

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Embedding Embryos in OCT Chamber

Reagents

* 50% Methanol in 1X PBS
* 1X PBS
* 30% Sucrose
	* Make 15mL of solution (bacteria may grwo over time)
	* 4.5gr of Sucrose in 1X PBS
* Tissue-Tek Cryomold
* OCT Compound
* Dry Ice

Prepping Embryos

  1. Embryos are stored in methanol in the freezer
  2. Pipette out the methanol in the embryo tube without sucking up the embryos
  3. Wash embryos with 50% methanol in 1X PBS for five minutes in mini shaker (set mini shaker settings to 190rpm)
  4. Three quick washes with 1X PBS (pipette out the old 1X PBS in between each wash)
  5. Wash with 1X PBS and leave in the mini shaker for 10 minutes
  6. Pipette out the 1X PBS
  7. Add 500uL of 30% Sucrose – sucrose displaces the water from your sample so upon freezing ice crystals do not form in your tissue
  8. Leave the embryos with 30% sucrose for 2 hours in the fridge

Putting Embryos In Cryomold

  1. Take a cyromold and use a gasket as a stencil to mark the middle of the cryomold. This will be the only area where you will want to place your embryos during cryosectioning
  2. Cut the bottom of a 200uL tip at a diagonal angle
  3. Take 100uL of the embryos from the bottom of the tube and place them in the center of the Cryomold
  4. Pipette out the remaining liquid with a new pipette tip (10uL tips are useful for this step)
  5. Fill the chamber with OCT Compound. Recommended to fill from the corners and not directly on top of the embryos
  6. Place the Cryomold on top of flat dry ice for at least 10 minutes
  7. Store Cryomolds with embryos in the freezer