CRF1&2 Receptor Binding Autoradiograpy (for primate tissue) : 1. Warm slides to room temperature 2. 2 min. fixation in 0.1% paraformaldehyde (rodent tissue does not need this step. Optional) 3. 2 X 15 min. rinse in receptor incubation buffer (to remove endogenous CRF) -bacitracin and aprotinin can be excluded in this step4. 2 hr. incubation with radioligand at room temp in incubation wells: Total CRF1&2 receptor binding: - 125I-sauvagine (0.2 nM; note: 0.1 nM can be used for big assays, but it requires longer exposure time to film and signal is not as “crisp”). [NEN/Dupont: catalog # NEX-306]. CRF2 receptor binding: - Add 1uM CP-154,526-1 from Pfizer (or any other specific CRF1 receptor ligand) to 125 I sauvagine in order to displace tracer from CRF1 receptors. Leftover binding will be specific CRF2 receptor binding. [Weigh 1mg CP154,526 with clean spatula*/1ml (50%EtOH/5mM HCl), MW=364.35; this solution will be 2,745 uM. Dilute 3.64 ul/10 ml 125I-sauvagine incubation buffer for 1uM]. *clean spatula with Acetic Acid/ dH2O/ Methanol Non-Specific Binding - Add 1uM cold sauvagine (MW= 4599.1; American Peptide Company, catalog # 347-11) to 125I sauvagine to displace binding to both CRF1 and CRF2, in order to determine non-specific binding. 5. 2 X 10 min rinse in 1XPBS + 1%BSA(pH 7.4) at 4C (shaking or stirring). (Change every 2 racks). 6. 2X dip in ddH2O at 4C 7. Dry slides inmediately with blow dryer on “cool” setting (aprox. 15 min). 8. Expose to Biomax MR Film for 80-90 hours Refs: DeSouza, et al. J. Neurosci. 5(12) 3189-3203. 1985 Primus, et al. Neuropsychopharm 17(5) 308-317. 1997 Recipes Receptor Incubation Buffer: 50mM Tris (7.88g Tris HCl/l ddH2O) 10mM MgCl2 (2.03g MgCl2/l) 2mM EGTA (760 mg EGTA/l) 0.1% BSA (1g BSA, fraction V/l) 142mg Bacitracin/L (use only tested Bacitracin!!! Some batches may ruin your assay; in case of doubt, do not put bacitracin). 10mg Aprotinin/l Bring volume to 1 liter with ddH2O, pH to 7.4 0.1% Paraformaldehyde: 300mL ddH2O 1g NaOH 3.5g NaOAc Heat to 60C, add 0.3g paraformaldehyde pH to 7.4 with glacial acetic acid