Remove coverslip from well plate using tweezers, wash with PBS three times to remove culture medium.
Immerse coverslip (cells face up) into 4% Paraformaldehyde for 10-20 min (close lid to prevent volatilisation). Wash with PBS three times.
Place coverslips onto filter paper (cells face up), remove liquid and allow to dry for 8-10 h.
Note: cell climbing slice can be stored at -20 ℃ for one week, to thaw slice wash with neutral PBS at room temperature for 10-15 min.
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 37 ℃ 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.