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Standard Staining procedure for cultured cells:

FIXING CELLS: 

(1) Take cells (12 well Transwells, coverslips, etc) from the tissue culture incubator.

(2) Rinse cells by 3x with cold Pre-FIX buffer (150 mM NaCl, 2 mM CaCl2, 2mM MgCl2, 10 mM HEPES, pH7.5, 0.02% NaN3). Use a pipet tip/vacuum to remove buffer from Transwell/dish as much as possible.  Do this quickly to prevent drying of cells. Proceed to next step immediately. 

(3) Fix cells with 1% Formaldehyde in Pre-FIX buffer for 1-2 hours. 


(4) Quench cells with Quenching buffer (50 mM Tris, 100 mM NaCl,  10 mM HEPES, pH7.5, 0.01% TX-100, 0.02% NaN3) for at least 2 hours.  Keep in refrigerator until use.


IMMUNOFLUORESCENCE DETECTION OF JUNCTIONAL PROTEINS: 

(1) Rinse cells 3X with IMF (IMmunoFluorescence) buffer:100 mM NaCl,  10 mM HEPES, pH7.5, 0.01% TX-100, 0.02% NaN3. 

(2) Add primary antibodies at final concentrations of 1-10 microgram per mL in IMF buffer.  Incubate 3 hours at room temperature or overnight at 4 degrees celsius. 

(3) Wash cells 4 times 15 min each in IMF buffer. 

(4) Add secondary antibodies at final concentrations of 1-10 microgram per mL in IMF buffer. Incubate 2 hours at room temperature. 

(5) Wash filters 4 times 15 min each in IMF buffer. 

(6) Optional: stain with phalloidin, Hoescht 33528, etc for 1 hour. Room Temperature.

(7)  Wash filters 4 times 15 min each in IMF buffer. 

(8) Fix cells with 1% Formaldehyde in IMF buffer for 30 min.  
 
(9) Quench cells with Quenching buffer (50 mM Tris, 100 mM NaCl,  10 mM HEPES, pH7.5, 0.01% TX-100, 0.02% NaN3) for 30 min.
 
(10) Mount filters with cells facing up. Place filter on a glass slide, add a drop of antifade reagent/mounting medium (ProLong Antifade Gold). Cover filter with a glass coverslip, let cure overnight before sealing. Room temperature in dark.

Vivian Tang Lab Notebook 2013