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.
