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Is gluteraldehyde fixation required because the immunogen was glutaldehye-linked to BSA? We would like to try this on sections from animals already perfused with 4% paraformaldehyde.
Asked on Nov 11 2004
Thank you for your enquiry. For Immunohistological studies with dopamine antibodies, fixation with glutaraldehyde is required. Please find below a protocol. Perfusion protocol for adult male Sprague-Dawley (weight around 0.5 kg) 1- The animals can be deeply anaesthetized for example with uerethane (0.1-1.5g/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 0.1M Phosphate buffer (PB), pH 7.2 ( in two minutes). b. 600 ml of cold 4% paraformaldehyde and 2% glutaraldehyde in 0.1M PB, pH 7.2 ( in ten minutes). c. Dissect out the brains and place in a solution of 4% paraformaldehyde in 0.1M PB, pH 7.2, at 4°C for twelve to sixteen hours. d. Before brain will be cut on a freezing microtome, we must include it in growing concentrations of sucrose (5 at 30 %). If you have any further questions then please do not hesitate to get back in touch with us.
Answered on Nov 15 2004