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Disclaimer text: All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunofluorescence staining
- Take out slides from -80 °C. Keep slides at RT for 1 h to airdry sections. Rinse slides in 0.1% PBST (0.1% Polyethylene glycol tert-octylphenyl ether in PBS; see Table of Materials) three times for 5 min each to wash out OCT and permeabilize sections.
- Optionally, perform antigen retrieval (optional).
- Preheat the citrate buffer (10 mM sodium citrate pH 6) in the staining dish with a steamer or water bath to 95–100 °C. Immerse the slides in the citrate buffer and incubate for 10 min.
- Take the staining dish out of the steamer or water bath and cool the slides at room temperature for 20 minutes or longer15.
NOTE: As alternatives, use Tris-EDTA buffer (10 mM Tris base, 1mM EDTA, 0.05% Tween 20, pH 9.0) or EDTA buffer (1 mM EDTA, 0.05% Tween 20, pH 8.0) for heat-induced antigen retrieval. Use a pressure cooker, microwave, or water bath for heat-induced antigen retrieval in addition to the hot steamer. An enzyme-induced antigen retrieval using trypsin or pepsin is another alternative. Optimize the concentration and treatment time of enzymatic retrieval to avoid damaging sections. Optimize the antigen retrieval method for each antibody/antigen combination.
- Incubate each slide with 200 μL of blocking solution (5% donkey serum diluted in 0.1% PBST) at RT for 30 min, then remove the blocking solution without rinse.
- Incubate each slide with 100 μL of primary antibody or antibodies diluted in blocking solution for 1 h at RT or O/N at 4 °C. Rinse slides with PBS three times for 10 min each at RT.
- Incubate each slide with 100 μL of secondary antibody diluted in blocking solution for 1 h at RT. Rinse slides in PBS three times for 10 min each at RT. Protect slides from light.
- Mount slides.
- Add two drops of anti-fade medium with DAPI (4', 6-diamidino-2-phenylindole) on the slide. Then cover with a coverslip.
- Store at 4 °C in the dark until ready to image.
NOTE: As an alternative, label the nuclei with DAPI or Hoechst 33324 dye diluted 1:2,000 in PBS at RT first, then mount with glycerol.