Immunohistochemistry Protocol for use with GPCR Polyclonal Antibodies from Proteimax

Note: The following protocol has been developed for use with GPCR Polyclonal Antibodies manufactured by Proteimax. Other antibodies have not been tested using this protocol. For additional assistance, please contact Technical Support.

Immunofluorescence

1. Wash three times 10 minutes with 0.1 M PBS, pH 7.4, shaking at room temperature.
2. Block non-specific binding by adding Diluent supplemented with 5% normal goat serum, overnight at 4°C (on a 60rpm rotator)
   Diluent:
   100 mL PBS 0.1 M pH=7.4
   300 µL Triton X-100
3. Add the primary antibody diluted in diluent supplemented with 5% normal goat serum. Incubate with primary antibody 20 to 48 hours at 4°C (on a 60 rpm rotator).
4. Wash three times 10 minutes with 0.1 M PBS, pH 7.4, shaking at room temperature.
5. Add the fluorescent labeled secondary diluted in Diluent supplemented with 5% normal goat serum. Incubate with secondary antibody one hour at room temperature (on a 60 rpm rotator) using the manufacturer’s recommended dilution.
6. Wash three times 10 minutes with 0.1 M PBS, pH 7.4, shaking at room temperature.
7. Put the material on the slice. Make sure to use buffer containing glycerol.

Immunoperoxidase

1. Wash three times 10 minutes with 0.1 M PBS, pH 7.4, shaking at room temperature.
2. To inhibit endogenous peroxidase – incubate with 0.3% hydrogen peroxide for 30 minutes at room temperature.
3. Wash three times 10 minutes with 0.1 M PBS, pH 7.4, shaking at room temperature. Wash until you can not see additional bubbles.
4. Block non-specific binding by adding Diluent supplemented with 5% normal goat serum, overnight at 4°C (on a 60 rpm rotator)
5. Add the primary antibody diluted in diluent supplemented with 3% normal goat serum. Incubate with primary antibody 20 to 48 hours at 4°C (on a 60 rpm rotator).
6. Wash three times 10 minutes with 0.1 M PBS, pH 7.4, shaking at room temperature.
7. Add biotinylated secondary antibody diluted in Diluent supplemented with 3% normal goat serum. Incubate with secondary antibody one hour at room temperature (on a 60 rpm rotator) using the manufacturer’s recommended dilution.
8. Wash three times 10 minutes with 0.1 M PBS, pH 7.4, shaking at room temperature.
9. Add the avidin-biotin-peroxidase complex (ABC Elite Vector Kit) – in 10 mL of 0.1 M PBS, pH 7.4, add 1 drop of A and B reagent. This solution should be prepared 15 to 30 minutes before incubation. Allow the solution to react for 45 minutes at room temperature (on a 60 rpm rotator).
10. Wash twice 10 minutes with 0.1 M PBS, pH 7.4, shaking at room temperature.
11. Wash twice 10 minutes with 0.05 M Tris-HCl, pH 7.6, shaking at room temperature.

    Developing:

12. Pre-incubate the slices in the developing solution (10 mL 0.05 M Tris-HCl, pH 7.6 and 5 mg DAB) for a short period of time.
13. In the beginning of the reaction: add 3 µL 30% H₂O₂.
14. The reaction should be controlled under the microscope, until the labeling has the desired color, approximately 2 to 10 minutes.
15. Stop the reaction by adding water or PBS.
16. Wash three times 10 minutes with 0.1 M PBS, pH 7.4, shaking at room temperature.
17. Put the material on the slice. Make sure to use buffer containing Permount or DPX.