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Immunohistochemistry Using “Extracellular” ATTO-Conjugated Primary Antibodies

Sacrifice and tissue processing:

1. Anesthetize rats with pentobarbital sodium (Pental).
2. Fix tissue/organ by transcardial perfusion, first with 50 ml of phosphate buffered saline (0.02 M PBS, pH 7.4) containing heparin (5 U/ml), then with 220 ml of ice-cold 4% paraformaldehyde in 0.1 M PBS, pH 7.5 containing 4% sucrose.
3. Cut tissue in coronal blocks and further fix by immersing in the same fixative.
4. Refrigerate for 1-2 hours.
5. Transfer tissue blocks to 12% sucrose in 0.1 M PBS.
6. Section tissue in a cryostat within 10 days.
7. Float tissue sections, 30 µm thick, in 0.1 M PBS and then preserve in a cryopreservation buffer preserve in a cryopreservation buffer (40% ethylene glycol and 1% polyvinylpyrrolidone in 0.1M potassium acetate buffer, pH 6.5) at -20°C.

Staining procedure:

1. Rinse floating sections in 0.02 M PBS, 2 x 5 minutes.
2. For light antigen retrieval, treat sections in 0.2 % hydrogen peroxide in 0.1 M phosphate buffer pH 7.3 containing 0.05% Triton X-100 for 25 minutes at room temperature.
3. Rinse sections in 0.02M PBS, 2x 5 minutes.
4. Incubate sections with the primary antibody diluted in a medium containing 0.05% Triton X-100, 0.05% Tween 20, 2% normal donkey serum (NDS), for 1 hour at room temperature.
The optimal dilution when using ATTO-labeled antibodies should be tested first with relatively low dilution e.g. 1:60 and increase the dilution to optimize signal to background ratio.
5. Refrigerate overnight.
6. Rinse sections in 0.02 M PBS, containing 2% NDS, 2 x 5 minutes.

Detection procedure using fluorescent microscopy:

Mounting, counter-staining, and cover-slipping:
1. Mount sections on glass slides from 0.02 M PBS, pH 7.0-7.4.
2. Allow to dry in a fume hood for 1 h.
3. Stain section on slides with DAPI (Molecular Probes), by placing DAPI solution on each slide, and covering the slide with a piece of parafilm to spread the solution evenly on the slide.
4. After 2 minutes, remove the parafilm and rinse the slide with 0.5 ml distilled deionized water using a pipette (repeat twice).
5. Let slides dry in a fume hood for 0.5 h.
6. Coverslip using the glue ImmuMount (Shandon, UK).
7. Leave slides to dry overnight in the dark.
8. Store at -18 ºC until confocal microscopy.
If the final result is negative, e.g. no staining, then extensive antigen retrieval is recommended.