Wash fresh tissue with PBS several times to remove blood. Remove connective tissue using tweezers and carve tissue into small pieces.
Prepare elongated tin foil. Add OCT embedding medium (to cover bottom part). Fix tissue and pour enough OCT embedding medium into mold. Using tweezers, place the bottom part of tin foil into liquid nitrogen for 1-2 min (until OCT embedding medium colour changes to white). Record tissue details with oil pen.
Pre-cool slicer box to -22 ℃. Pre-cool detector to -24 ℃. Ensure completeness and smoothness of blade.
Open tin foil and put the tissue into pallet; fix on detector. Adjust and slice tissue quickly and carefully to 5 um thickness. Use cover slip to take sliced tissue. Allow to dry.
Fix frozen section by immersion into 4% Paraformaldehyde for 10 min. Wash with PBS several times. Allow to dry. Store at -20 ℃ for two months.
Citrate buffer (pH 6.0)
Compound digest solution
1% Triton X-100
5% BSA blocking solution/goat serum
Antifade mounting media
Prepare a 1 in 10 solution of 30% H2O2 in distilled water (mix one part 30% H2O2 with nine parts distilled water).
Immerse dewaxed paraffin section into solution at room temperature for 10 min.
Wash with distilled water for several min.
Prepare a 1 in 50 solution of H2O2 in methanol (mix one part H2O2 with 49 parts methanol).
Immerse frozen section and cell slice into solution at room temperature for 10 min.
Wash with distilled water for 1 min, 3 times.
Heat repair: Immerse the paraffin sections into citrate buffer (pH 6.0), microwave until boiling then cut off the power, leave in microwave for 10 min. Repeat twice. Cool at room temperature and wash twice by immersing in distilled water.
Enzyme digestion: use filter paper to dry extra distilled water upon frozen sections or cell slice. Add compound digest solution and incubate at room temperature for 3-5 min. Wash with PBS for 5 min, 3 times. With regards to cell slice, it is recommended to add 0.1% Triton and incubate for 10 min before enzyme digestion. Then wash with PBS for 10 min, 3 times.
Add 5% BSA blocking solution or normal goat serum and incubate at 37oC for 30 min with shaking.
Adding primary/secondary antibody
Dilute primary antibody in blocking buffer. Incubate at 37oC
Wash with PBS for 20 min, 2 times. Add labelled secondary antibody and incubate at room temperature in the dark for 1-2h.
Note: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.
Wash with PBS for 20 min, 2 times. Add SABC reagents and incubate at 37 ℃ for 30 min. Wash with PBS for 20 min, 3 times.
Prepare solution by mixing 1 ml distilled water with one drop of Reagent A, B, C.
Add solution to slice. Incubate at room temperature as per instructions.
In preparation for counterstaining; with IB4/DAPI or GFAP/DAPI, incubate the sections in PBS with Ca2+, Mg2+, and Mn2+ and 0.1% Triton X-100 for 15 minutes. Rinse the slides through a quick change of PBS.
Incubate slides in 1:10,000 DAPI for 30 min. Wash slides in PBS for 2 min, 3 times.
Prepare antifade mounting media according to directions. Allow the slides to dry approximately ¾ to completion. Place the coverslip taking care to remove any bubbles formed beneath the coverslips.