a. Precool ThermalTray and Coolsink by filling large thermal safe container with dry ice and covering the ThermalTray and CoolSink, give 10-15 minutes for the trays to cool. Be cautious and wear the appropriate PPE when handling dry ice. (Used in Freezing Block step)
b. Using a 6-well plate, or other small containers such as medium weigh boats, fill the 1st with 100% 1X PBS, the 2nd with 1:1 PBS & OCT, and the third with 100% OCT.
c. Take tissue sample and place in 1st container with 100% 1X PBS. Using forceps gently move the tissue around in the PBS for around 30 seconds to 1 minute, removing any sucrose or other potential debris. Do not leave tissue in PBS for long periods of time as it will undo the sucrose cryoprotection.
d. Transfer tissue to 2nd container and repeat gentle movement in 1:1 PBS & OCT solution for 30 seconds to 1 minute.
e. Transfer tissue to 3rd container containing 100% OCT and gently move the tissue around for 30 seconds to 1 minute, ensuring that tissue is fully coated in OCT. Use extra care when handling fragile tissue as the increased viscosity of OCT can cause tissue to separate or tear.
f. Replace the reagents in the containers every 3 to 5 pieces of tissue, or when they appear visibly dirty. Failure to use fresh reagents may result in cross-contamination between samples.