Procedure for Free-Floating Sections

Free-floating sections (40 xm) of paraformaldehyde-fixed tissues are rinsed three times for 5 min each in 0.1 M sodium phosphate buffer (pH 7.4) (Jiao et al., 1999). They are transferred to 10-15 mM sodium citrate buffer (pH 8.5-9.0) preheated in a water bath kept in a conventional oven at 80°C for 30 min. The sections are allowed to remain in this buffer for 30 min to cool to room temperature. Following rinsing three times for 5 min each in the same buffer, the sections are treated by immersion in 0.3-3% nonfat dry milk in 0.1 % sodium azide for 30-60 min. The sections are then incubated in the primary antibody, diluted with a mixture of 0.3% Triton X-100, 0.01% sodium azide, 0.1 M sodium phosphate buffer (pH 7.4) (PBX), and 5% normal horse serum for 72 hr at 4°C under constant agitation. 

The sections are rinsed in the same buffer and incubated for 90 min in secondary antiserum (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted 1 50 with PBX-5% normal horse serum. They are rinsed in 0.1 M sodium phosphate buffer and then incubated for 1 hr in peroxidase antiperoxidase (PAP), diluted 1 100 with PBX. The sections are rinsed three times for 5 min each in the same buffer followed by distilled water rinses. They are incubated for 10 min in 50 ml of 0.05 M imidazole/0.05 M cacodylate buffer (pH 7.2) containing 50 mg of DAB. This is followed by an additional 10 min incubation after adding 200 xl H202. All incubations are carried out under constant agitation. The sections are washed in distilled water, placed in the same buffer, mounted onto gelatin-coated slides, dried, dehydrated, and cover-slipped with Permount. Note that the ABC procedure can be used instead of the PAP procedure. 

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