contributed by Patricia Tsao
This protocols allows the use of fluorescence microscopy to visualize epitope-tagged receptors expressed by stable adherent cell lines. In contrast to the permeabilized cell protocol, this protocol where antibody is "fed" to live unpermeabalized cells will only visualize receptors that started out on the surface of the cell.
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 :
1. Using a 6 well tissue culture plate, add 2mL of media to each well to be used.
2. Equilibrate media in incubator (~30-45').
3. Add coverslips. Equilibrate in incubator (~ 25').
4. Add primary antibody at 1:500 to 1:1000 dilution to media for 30'. Example antibodies: M1 mouse anti-Flag antibody (Sigma, Covance) (Ca 2 sensitive) 1:350-1:500; 12CA5 mouse anti-HA (Roche)1:1000 polyclonal
5. Treat cells. Typical incubation is approximately 25'.
6. Quickly aspirate media and add fixative (~ 2 mL/well). Fix for 15-20'.
7. Wash 3x.
8. Aspirate the final wash, taking care to dry the edges of the coverslip well. Add Blotto to only the coverslip (~ 50 uL) and incubate 20'. VARIATION: Some people prefer transferring 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.
9. 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.
10. Wash 3x.
11. 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.
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