Cool snap rs photometrics camera

After 24 h of treatment with 100 nM bexarotene or DMSO, BLECs were washed with pH 7.4 phosphate buffered saline-calcium-and magnesium-free solution (PBS-CMF; 8.0 g/L NaCl, 0.2 g/L KCl, 0.2 g/L, KH 2 PO 4 et 2.87 g/L Na 2 HPO 4 -12H 2 O) once and then fixed in 4% paraformaldehyde for 10 min at room temperature (RT). After three washes in PBS-CMF, filters were cut free from the plastic insert and the cells were rinsed three times in PBS and permeabilized with Triton X-100 (0.1% (w/v)) in PBS-CMF for 10 min at RT. Following a 30-min incubation in PBS-CMF supplemented with 10% (v/v) normal goat serum (NGS), cells were incubated for 1 h with the primary antibody at RT (anti-ZO1 (red) or anti-Occludin (green), Invitrogen, Cergy-Pontoise, France). After 3 washes in PBS-CMF supplemented with 2% NGS, preparations were incubated with the secondary antibody for 30 min at RT. Nuclei were stained using Hoechst reagent (blue). Cells were mounted using Mowiol (Sigma-Aldrich) containing 1,4-diazabicyclo[2.2.2]octane (Sigma-Aldrich) as an anti-quenching agent. Images were collected using a Cool SNAP RS Photometrics camera (Leica Microsystems) and processed using Adobe Photoshop software (version 5.5, Adobe Systems, San Jose, CA, USA).

MBCECs were rinsed twice with warmed CMF-PBS and then fixed for 1 min in cold methanol/ acetone (v/v). After three washes with CMF-PBS, the filters were cut from the plastic insert and (to avoid nonspecific binding) incubated for 30 min in CMF-PBS with 10% normal goat serum. The cells were then incubated with the primary antibody (anti-ZO-1; dilution: 1/200; anti-claudin 5 (dilution: 1/100; both from Invitrogen, Cergy-Pontoise, France) diluted in CMF-PBS supplemented with 2% NGS for 1 h. After three washes in CMF-PBS with 2% NGS, the cells were incubated in the diluted secondary antibody (AlexaFluor 488/568; Invitrogen) for 60 min. Nuclei were stained with Hoechst 33258 reagent. Lastly, after a wash with CMF-PBS with 2% NGS followed by three washes with CMF-PBS, the cells were mounted using Mowiol reagent containing Dabco (Sigma-Aldrich). A Cool SNAP RS Photometrics camera (Leica Microsystems) was used to acquire the images, which were then processed using Adobe Photoshop software (Adobe Systems, San Jose, CA, USA).