Ab31721

Western blotting of ileum and colon were performed as previously described^{61 (link)}. The primary antibodies against claudin 1 (1:1000 dilution, ab15098), occludin (1:1000 dilution, ab31721), and ZO-1 (1:1000 dilution, abG041) were purchased from Abcam.

Lungs were collected and fixed in 4% buffered formaldehyde in PBS pH 7.2–7.4 (Bio Lab, Jerusalem, Israel) for 2 weeks. Sections of 5 µm were prepared after paraffin embedding using an RM 2255 microtome (Leica, Nussloch, Germany). Antigen retrieval was performed by incubation in Target Retrieval Solution (S1700, DAKO, Carpinteria, CA, USA, 30 min, 95 °C). After blocking in 5% BSA in PBS, slides were incubated (overnight, 4 °C) with purified anti-CD31 (390, Biolegend, San Diego, CA, USA), proSPC (Millipore, Temecula, CA, USA), podoplanin (T1α, 8.1.1, Biolegend), VE-cadherin (ab33168), claudin 5 (ab15106), connexin 43 (ab117843), occludin (ab31721) or claudin 18 (ab203563) (Abcam, Cambridge, MA, USA). Alexa Fluor 594- or 488-coupled donkey anti-rabbit or Alexa Fluor 594-coupled goat anti-Armenian hamster antibodies were used for detection (Molecular probes®, Thermo Fisher Scientific, Carlsbad, CA, USA). For nuclear staining, slides were mounted with Prolong® Gold antifade reagent containing DAPI (Molecular probes®, Thermo Fisher Scientific, Carlsbad, CA, USA). Analysis was performed using an LSM 710 confocal scanning microscope (Zeiss, Jena, Germany) equipped with the following lasers: argon multiline (458/488/514 nm), diode 405 nm, DPSS 561 nm and helium-neon 633 nm.

Caco-2 cells were cultured using high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% nonessential amino acids, 4.5 g/L D-glucose, 110 mg/L sodium pyruvate, and 1% antibiotic cocktail. Cells were grown until confluence, on cover slips coated with collagen, and then treated with 2% DSS or a combination of 2% DSS and 100 nM Pld2 inhibitor (VU0364739.HCl, Tocris). The control group received either DMSO or PLD2 inhibitor alone without DSS. After 12 hours of DSS treatment, the cells were fixed using 4% paraformaldehyde for 20 min at 25 °C. Cells were then washed with PBS and permeabilized with 0.1% Triton X-100 diluted in PBS for 15 min at room temperature. After permeabilization, the cells were washed with PBS and incubated with 5% goat serum for 1 hour at room temperature, followed by incubation with primary antibody against occludin (ab31721, rabbit anti-occludin, Abcam) overnight at 4 °C. Alexa 594-labelled anti-rabbit IgG was used as the secondary antibody. Nuclear staining was performed using 4″,6′-diaminido-2-phenylindole. The sections were analysed using digital micrographs and an LSM 700 ZEISS laser scanning confocal microscope (Carl Zeiss, Jena, Germany).