1. Insert tissue aliquot in a 50 ml tube
2. Freeze the tube in liquid nitrogen (5 minutes) then move to -80C freezer.
ii. Fixed frozen OCT-embedded:
1. Immerse the tissue aliquot (max 2 cm x 2 cm x 1 cm) in a 50 ml tube with 4% paraformaldehyde (Electron Microscopy Sciences #15710) diluted in DPBS. Use a ratio tissue/fixative of 1:10 and store at 4C for 16 hours.
2. Discard the fixative, rinse with DPBS.
3. Wash twice for 5’ by filling the tube with DPBS.
4. Fill the tube with 35% sucrose solution and keep 24h at 4C.
5. Embed in O.C.T. compound.
6. Freeze the block by placing on top of metal cassettes immersed in liquid nitrogen in a closed Styrofoam box (the block does not touch directly the liquid nitrogen)
7. After 10’ move to -80C freezer.
iii. Fixed paraffin-embedded:
1. Immerse the tissue aliquot (max 2 cm x 2 cm x 1 cm) in a 50 ml tube with 4% paraformaldehyde (Electron Microscopy Sciences #15710) diluted in DPBS. Use a ratio tissue/fixative of 1:10 and store at 4C for 16 hours.
2. Discard the fixative, rinse with H20.
3. Wash twice for 60’ by filling the tube with H20.
4. Replace H20 with 50% ethanol and incubate 3 hours
5. Replace with 70% ethanol and proceed with standard embedding protocol or store in 70% ethanol at 4C.