- Repeat wash procedure in 1PBS containing 0.18% Tween
20 at least 3–5 times and then visualize the immunoreactive
bands. - A capable imaging system and dedicated image analysis soft-
ware can be employed to detect the signals and quantify the
density ratios of immunoreactive bands.
3.5 Fluorescence
Staining, Confocal
Laser Scanning
Microscopy (CLSM),
and Stimulated
Emission Depletion
(STED) Super-
Resolution Microscopy
- Caco-2 cell monolayers are allowed to age 7–10 days on glass
coverslips(13mmdiameter withathicknessof0.170.01mm)
and then exposed to the desired concentration of 3O-C 12 -HSL
or 0.6% DMSO as a diluent control for the desired period of
time at 37C. - Following exposure to quorum sensing molecule(s) or diluent
control, rinse coverslips twice with 1PBS pH 7.3 and fix cells
with 3% paraformaldehyde in 1PBS for 20 min at room
temperature. Again, rinse coverslips in 1PBS following fixa-
tion and then permeabilize the cells in 0.18% Triton X-100 in
1 PBS for 5 min at room temperature. Rinse cells on coverslip
with 1PBS when completed. - Block nonspecific staining with blocking buffer (1PBS sup-
plemented with 1% BSA and 10 mM glycine) for 1 h at room
temperature. Wash cells on coverslip with 1 PBS when
completed. - Dilute the desired primary Abs in blocking buffer (working
dilutions should be empirically determined for each respective
Ab) and then apply to coverslips and incubate for 1 h at room
temperature (or overnight at 4C) in a humid chamber. As an
example, we have used anti-ZO-3 Abs, anti-JAM-A antibodies,
or anti-IQGAP1 Abs (Figs.2b and 3). - Following primary Ab(s) incubation, coverslips are rinsed with
1 PBS and the Alexa Fluor 488-conjugated goat anti-rabbit
Abs, Alexa Fluor 568-conjugated goat anti-mouse Abs, or Atto
647 N (fluorophore for STED)-conjugated goat anti-mouse
secondary Abs are added to the coverslips and incubated in
humid chamber for 1 h at room temperature in the dark.
Appropriate dilutions of detection Abs should be empirically
determined. - If desired, cell nuclei can be stained with DAPI (4^0 ,6-Diami-
dino-2-Phenylindole, Dihydrochloride). - Finally, after secondary Ab(s) incubation, coverslips are rinsed
with 1PBS a final time and then mounted onto glass slides
with Prolong®Gold Antifade Reagent (Life Technologies,
Eugene, OR).
222 Jake Everett et al.