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Application Notes
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Example of cytochemistry application Perfusion protocol for adult male sprague dawley (weight around 0.5kg):1- The animals can be deeply anesthetized with for example urethane (0.5-1.5g/kg, intraperitoneal). 2- Heparinized, and perfused via the ascending aorta with 100ml of cold physiologic saline (0.9% NaCl) and with the following fixative solution:a) 300ml of cold 4% paraformaldehyde and 2% glutaraldehyde in 0.1M phosphate-buffer (PB), pH 7.2 (two minutes). b) 600ml of cold 4% paraformaldehyde and 2% glutaraldehyde in 0.1M 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 (0.1M, 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 0.15M 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 (1h30min) and overnight at 4ºC in the same mixed solution containing L-Arginine antiserum (diluted 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 1h 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). Gemacbio sells the anti-conjugated Citrulline and anti-conjugated Agmatine antibodies raised in rabbit: used together, these tools could be helpful for immunocytochemistry double labelling. 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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