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Application Notes
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Immunohistochemistry Perfusion protocol for Adult male Sprague Dawley (weight around 0.5 kg):1-The animals can be deeply anaesthetized for example with urethane (0.5-1.5g/kg, intraperitoneal). 2-Heparinized, and perfused via the ascending aorta with 100 ml of cold physiologic saline (0.9% NaCl) and with the following fixative solution:a) 300 ml of cold 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate-buffer (PB), pH 7.2, (two minutes). b) 600 ml of cold 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate-buffer (PB), pH 7.2, (ten minutes). c) Dissect out the brains and place in a solution of 4% paraformaldehyde in 0.1M PB, pH 7.2, at 4ºC for twelve to sixteen hours. d) Before the brains will be cut on a freezing microtome, we must include the brain in growing concentrations of sucrose (a first bain of 5% of sucrose in PBS until the brains sank), after that we will repeat the same process in a solution with a higher level of sucrose (10%), 20%, 25% and finally 30%. Around 50 µm-thick serial sections will be obtained, kept at 4º C in PBS (0.1 M, pH 7.2) and processed for immunostaining. Example of immunohistochemical protocol:1-In order to avoid possible interference with endogenous peroxidase, free-floating sections will be treated with distilled water containing NH 3 (20%), H 2 O 2 (30%) and NaOH (1%) for 20 min (other method is using a solution with 33% of H 2 O 2 and 66% of methanol). 2-Then, wash the sections for 20 min in 0.15 M phosphate-buffered saline (PBS) (pH 7.2) 3-Pre-incubate for 30 min in PBS containing 10% of normal horse serum and 0.3% of Triton X-100 (mixed solution). 4-Incubate at room temperature (1h 30min) and overnight at 4º C in the same mixed solution containing anti-conjugated Adrenaline antibodies (diluted 1/1,000-1,5000 as recommended dilution). 5-Then, the sections will be wash in PBS (30 min). 6-After that we will incubate for 60 min at room temperature with biotinylated anti-rabbit immunogammaglobulin (Vector) diluted 1/200 in PBS. 7-Wash during 30 min with PBS. 8-Sections will be incubated for 1 h with a 1/100 diluted avidin-biotin-peroxidase complex (Vectastain). 9-After that we will wash the sections in PBS (30 min) 10-Wash with Tris-HCl buffer (pH 7.6)(10 min). 11-The tissue-bound peroxidase will be developed with H 2 O 2 using 3, 3' diaminobenzidine as chromogen. 12-Finally the sections will be rinsed with PBS and coverslipped with PBS/Glycerol (1/1). Recommended dilutions for Immunocytochemistry (1/1,000-1/5,000) Recommended dilutions for Western Blot (1/1,000-1/2,000)
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