Wash fresh tissue with PBS several times to remove blood. Remove connective tissue using tweezers and carve tissue into small pieces. Immerse into 4% Paraformaldehyde for 8-10 min.
Fix tissue by immersion in pre-cooled 4% Paraformaldehyde (4oC) (20 times the volume of the tissue sample) for 6-7 h.
Note: Over-fixation can mask the epitope. Under-fixation can lead to edge staining.Note: 4% Paraformaldehyde can be substituted by Neutral Buffered Formalin (10%) and left for 24-48h.
Wash with PBS for 1 min, 3 times.
Dehydrate tissue by immersion into ethanol (80% ethanol for 1 h, then 90% ethanol for 1 h, then 95% ethanol for 1 h, 3 times and finally 100% ethanol for 1 h, 3 times (total time 8 h)). Carry out dehydration at 4oC.
Clear the tissue by immersion in dimethylbenzene at room temperature for 30 min, 3 times.
Immerse the tissue into prepared liquid paraffin at 58oC-60oC for 2 h, 2 times.
To embed in paraffin block: pour liquid paraffin pre-heated to 60oC into mold, place tissue into the block as soon as possible, incubate at room temperature until the paraffin sets. Store at 4oC until sectioning.
To slice paraffin sections: fix paraffin section on slicer, slice extra paraffin and adjust slicer to ensure paraffin and blade are parallel. Slice paraffin section carefully. Cut 4-6 mm slices.
Incubate sliced paraffin section in a water bath containing distilled water at 40oC - 50oC to unfold. Prepare glass slide by immersion into APES or Poly-Lysine. Dip out paraffin section with prepared glass slide. Incubate slide at 37oC
Dry the paraffin section by incubating for 2h at 60oC.
Xylene (90%, 95%, 100%)
Ethanol (90%, 95%, 100%)
Citrate buffer (pH 6.0)
Compound digest solution
1% Triton X-100
5% BSA blocking solution/goat serum
Antifade mounting media
Prepare three bottles of 90%, 95% and 100% xylene and three bottles of 90%, 95% and 100% ethanol.
Immerse paraffin sections into three bottles of xylene orderly (concentration from low to high) for 7 min each. Then immerse into ethanol orderly at room temperature (same order as xylene) for 7 min each.
Wash with water to remove ethanol.
Note: dewaxing should be done in a fume hood at room temperature. If the temperature is below 18 ℃ it is recommended to dewax at 50 ℃.
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.