Staining for total receptors

This protocol will visualize both internalized receptors that were once on the plasma membrane and receptors in the biosynthetic pathway. This protocol was specifically written for visualizing agonist-induced internalization of receptors.

Materials:

Wash Solution -- Total Volume 2 liters; Maniatus TBS with 1mM CaCl2, 16g NaCl, 6g Tris base, 0.4g KCl, 0.294g CaCl2 . 2H2O, pH 7.4 with conc. HCl.

Blotto -- Total Volume 10mL, 0.3g dry milk (3%), 100ul 10% Triton X-100 (0.1%), 100ul 100mM CaCl2 (1mM), 500ul 1M Tris-Cl pH 7.5 (50mM).

Fixative -- approximately 2ml per coverslip - 37% formaldehyde diluted 1:10 with PBS (150mM NaCl, 10mM NaPi, pH 7.4)

Procedure :

Tissue Culture

Plate cells onto 18 mm square coverslips. Pretreatment of coverslip with polylysine helps cell remain adhered to the coverslip. Don't let the cells get too confluent, or else the entire "sheet" of cells will come off the coverslips during the washes.

1. Using a 6 well tissue culture plate, add 2 ml of media to each well.

2. Equilibrate media in incubator (~30-45').

3. Add coverslips. Equilibrate in incubator (~ 25 min). NOTE: It is preferable to perform both equilibration steps; however, it is not necessary.

4. Treat cells with agonist. (typically 5-25').

5. Quickly aspirate media and add fixative (~ 2 ml/well). Fix for 15-20'.

6. Wash 3x.

7. Aspirate the final wash, taking care to dry the edges of the coverslip well. Add Blotto to only the coverslip (~ 50uL) and incubate 20 minutes. NOTE: We only apply Blotto directly onto the coverslip in order to reduce the volume required to process each coverslip. VARIATION: Some people transfer the coverslip to a sheet of parafilm in a tissue culture dish with a small amount of wet whatman inside. The parafilm helps the antibody solution "bead up" on the coverslip, and the wet whatman paper helps prevent the antibody from drying up.

8. Aspirate Blotto, and apply primary Ab only to the coverslip. Incubate 45'. Dilute antibody with Blotto. Some typical dilutions are listed below: monoclonal M1 mouse anti-Flag antibody (Sigma, Covance) (Ca+2 sensitive) 1:350-1:500; polyclonal 12CA5 mouse anti-HA (Roche)1:1000.

9. Wash 3x.

10. Second Blotto block as above, 5'.

11. Aspirate Blotto, and apply secondary Ab. Incubate 20' in the dark. Dilute antibody with Blotto. Cy3 donkey anti-mouse (Jackson Immunoresearch) 1:500, FITC goat anti-mouse 1:500, TexasRed goat anti-mouse 1:500.

12. Wash 3x.

13. Wet mount with anti-fade solution, i.e. VectaShield (Vector Labs) or Citifluor (Ted Pella). Put small drop of mounting media on glass slide. Use forceps to pick up coverslip and aspirate any excess liquid. Place coverslip cell-side down onto glass slides. Aspirate any excess mounting media from edges of coverslip. Seal edges with nail polish.

References:

Jeffrey L. Benovic (Editor). Regulation of G Protein-Coupled Receptor Function and Expression (Receptor Biochemistry and Methodology).

Epitope Tagging, Basic Laboratory Methods. PDF manual from Roche website.