After blocking in IFA buffer (PBS containing 1% bovine serum albumin, 0.1% Tween 20), the coverslips were incubated with the primary antibody in IFA buffer followed by incubation in IFA buffer containing 0.5 μg ml−1 Hoechst 33342 and the secondary antibody conjugated to Texas red (Vector Laboratories).
The coverslips were incubated in blocking buffer (PBS containing 0.5% saponin and 1% BSA) for 30 min and then sequentially exposed to a mouse anti-BrdU antibody (diluted 1/1400, Cell Signaling) for 1 h and to a goat anti-mouse Alexa Fluor 555 antibody (diluted 1/400, Invitrogen) for another 1 h.
Cells on coverslips were incubated overnight in blocking buffer of PBS containing 10% (w/v) BSA and 0.15% (v/v) Tween-20.
Cells grown on glass coverslips were incubated for 10 min at 37 °C in incubation buffer containing 10 μM dihydroethidium, washed and incubated for further 30 min with buffer containing 1 μM A23187.
After rinsing in phosphate buffer saline- 0.1% Tween (PBST, 1X) the coverslips were incubated with anti-mouse FITC conjugated/anti-rabbit Alexa flour®594 conjugated secondary antibody for 45 min at 37 °C in the dark.
Coverslips were incubated 1 hr in blocking buffer (0.5% triton X-100, 1% BSA, in PBS) prior to antibody staining.
To extract fixed samples with detergent, fixed coverslips were incubated for 2 hr in buffer containing 1% Triton X-100 and 0.1% saponin.
Coverslips were incubated for 30 min in blocking buffer (DMEM, 10% FCS) and treated as described below.
The biofilm-coated coverslips were incubated overnight at 4°C in fixation buffer (4% paraformaldehyde, 2.5% glutaraldehyde, and 2 mM CaCl2 in 0.2 M cacodylate buffer, pH 7.2), washed twice with 0.1 M cacodylate buffer (pH 7.0), and postfixed for 90 min at room temperature in 1% osmic acid containing 2 mM potassium ferrocyanide and 6% sucrose in 0.1 M cacodylate buffer (pH 7.0).
For cell permeabilization, coverslips were incubated for 7 min in cold Digitonin Assay Buffer supplemented with 30 μg/ml digitonin and protease inhibitors (10 μg/ml each of leupeptin, pepstatin A, bestatin, aprotinin, AEBSF, and E-64).
