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 4C (shaking or stirring).
(Change every 2 racks).
6. 2X dip in ddH2O at 4C
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 60C, add 0.3g paraformaldehyde
pH to 7.4 with glacial acetic acid