1.Embed fresh tissues carefully in OCT in plastic mold, being careful not to trap air bubbles surrounding the tissue. Freeze tissue by setting mold on top of liquid nitrogen until 70-80% of the block turns white. Then, put block on top of dry ice. Frozen blocks may be stored at -80C for long-term storage.
2.For cutting step, mount the frozen block on the cryostat holder. Never, at any point, let the tissue warm up to temperatures above -15C.
3.Allow frozen blocks to equilibrate in the cryostat chamber for about 5 minutes. Cut 6-8 um sections. The best sections are usually obtained when the block temperature is around minus 18 to minus 20C.
4.Let the sections dry for at least 30 minutes at room temperature. Note: Upon drying, tissues can be stored at 4C for a few days; for longer-term storage up to a few months, store at -80C.
5.Fix the sections by immersing in acetone jar (or other appropriate fixatives) for 1-2 minutes at room temperature, and let air-dry. Carefully draw boundaries of sections using a PAP Pen and let it dry for a few minutes.
6.Add primary antibodies (diluted in TBS, pH 7.4, 2.5% normal serum) directly onto the sections. Add in big droplets to cover the entire tissue at once to prevent fracturing of the sections due to the surface tension of the solution. Incubate in a chamber for at least one hour at room temperature. Note: Once the sections are re-hydrated with TBS, never let the tissue dry out again (this will ruin the tissue architecture).
7.Wash the sections gently in TBS for 3-5 minutes and then in 2.5% serum solution for another 3-5 minutes.
8.If using biotinylated primary antibodies, skip the following 2 steps (8 and 9) and move to step 10.
9.If using purified primary antibodies, continue the protocol at step 8.