Y10b on Vibratome sections

Fixation.

Transcardial perfusion with 4% phosphate buffered paraformaldehyde, pH 7.4. Decapitate animal, expose brain by removing cranium, then immerse in 4% paraformaldehyde for overnight at 4° C.

Rinse in phosphate buffered saline (PBS) then vibratome at 30 - 40 µm, collecting sections in a one in four series.

Immunocytochemistry.

Rinse sections in PBS 3 times for 30 min. each.

All immunoreagents were diluted in 0.1% porcine gelatin, electrophoresis grade (Sigma, G-8150) dissolved in PBS by heating. All washes were for 3 times, 10 min. each in PBS, unless otherwise noted. All reactions were performed with free-floating sections in 24-well tissue culture plates

1. Incubate sections for 40 min. with 4% normal horse serum;

2. Withdraw normal serum with a pipette and replace with Y10b supernatant diluted with 0.1% gelatin, for 18-20 hrs. at 4° C, then wash. A dilution of 1/500 works for chicken brainstem, 1/1000 for gerbil;

3. Incubate for 60 min. with 1/400 biotinylated horse anti-mouse IgG (Vector Laboratories), at room temperature, then wash;

4. Incubate for 60 min. with 1/5 dilution of ABC reagent (Vector Laboratories, Vectastain Elite Standard Peroxidase Kit) - add 9 µl each of Reagent A and Reagent B per 1 ml of 0.1% gelatin, allow complexes to form for 30 min., then dilute 5-fold with 0.1% gelatin;

5. After washing, incubate 10 min. with 0.375 mg/ml DAB and 0.1% H2O2 in .1 M Tris-HCl buffer, pH 7.6.

Mounting.

Float labeled sections in a dish of PBS and arrange onto chrome alum subbed slides. Dry overnight, rehydrate and counterstain if desired, then dehydrate through graded ethanols, clear in xylene and coverslip with DPX.