Zo 1 antibody

Human omental mesothelial cells were plated at > 95% confluence in uncoated and Matrigel-coated 24-well plates and were allowed to adhere. Thereafter, culture media were removed, and cells were incubated with 0.5 mL normal saline or LRS at 37°C for 0, 5, 10, 15, 30, and 60 minutes with gentle shaking at 50 rpm. Following incubation, cells that remained adhered to plates were fixed in 4% paraformaldehyde and were stained with crystal violet solution. Adhered cells were quantified by solubilizing crystal violet dye in 10% acetic acid and reading absorbance at 570 nm using a Spark microplate reader. In other experiments, mesothelial cells were plated in 4-well Permanox chamber slides (Sigma-Aldrich) and were then incubated with normal saline or LRS as above. Cells that remained adhered were fixed in 4% paraformaldehyde and permeabilized by incubation in 0.2% Triton X-100 in blocking buffer for 10 minutes. Cells were then stained with ZO-1 antibody (#33-9100, Invitrogen) at 4°C for 16 hours, washed 3 times with PBS, and incubated with fluorochrome-conjugated secondary antibody for 1 hour. Concentrations of antibodies used are listed in Supplemental Table 2. Slides were then washed 3 times with PBS, stained with DAPI, and reviewed under a Nikon 80i fluorescence microscope.

Fura-2-acetoxymethyl ester (Fura-2-AM) and 2′,7′-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein- (BCECF-) AM were purchased from Teflabs (Austin, TX). 3-Aminobenzamide (3-AB), histamine, LPS from Pseudomonas aeruginosa serotype 10, rolipram, carbamyl choline chloride (carbachol), isoproterenol, hydrogen peroxide, trypsin inhibitor, sodium pyruvate, bovine serum albumin (BSA), β-actin antibody, and all other chemicals not mentioned here were purchased from Sigma. Phosphate-buffered saline (PBS), Pluronic F-127 (20% in DMSO), goat serum, E-cadherin antibody, ZO-1 antibody, PARP-1 antibody, and MQAE dye were purchased from Invitrogen (Carlsbad, CA). PDE4 and caspase-1 antibodies were purchased from Fabgennix (Frisco, TX) and Abcam (Cambridge, MA), respectively. Collagenase P was purchased from Roche (Basel, Switzerland).

Fura2-acetoxymethyl ester (Fura2-AM) and 2′,7′-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-AM were purchased from Teflabs (Austin, TX). LPS from P. gingivalis, histamine, Dexmedetomidine hydrochloride (Dex), isoproterenol, carbamyl choline chloride (CCh), yohimbine (YOH), bumetanide (Bumeta), and all other chemicals not mentioned here were purchased from Sigma Aldrich (St. Louis, MO). Pluronic F-127 (20% in DMSO) and ZO-1 antibody were purchased from Invitrogen (Carlsbad, CA). PDE4 was purchased from Fabgennix (Frisco, TX), and IL-6 and caspase-1 antibodies were purchased from Abcam (Cambridge, MA). The α2_A-adrenergic receptor antibody was purchased from Santacruz Inc. (Santa Cruz, CA). Collagenase P was purchased from Roche (Basel, Switzerland). The HA-tagged human AE2, pCMV6-AC-mKate-SLC26A6, and empty vectors were kindly provided by Dr. Shmuel Muallem (National Institutes of Health, Bethesda, MD, USA).

For immunostaining 3D MDCK cells in 30 µl blobs of Cultrex® 3D Culture Matrix™ BME (3445-005-01, Trevigen, Gaithesburg, Maryland, USA) were cultured in normoxic or hypoxic conditions for 6 days, 2D MDCK cells were cultured in normoxic conditions for 24 h. The immunostaining procedure was performed as described previously^{24 (link)}. The following antibodies were used: podocalyxin antibody (3F2/D8 cell line, Developmental Studies Hybridoma Bank, Iowa City, Iowa, USA), E-cadherin antibody (rr1, 1:100, Developmental Studies Hybridoma Bank, Iowa City, Iowa, USA) and ZO-1 antibody (339100, 1:200, Invitrogen, Carlsbad, California, USA), all corresponding secondary antibodies were purchased from Abcam (Cambridge, UK), TRITC-phalloidin (P1951, 1:500, Sigma Alrich, St. Louis, Missouri, USA) and DAPI (D9542, 1:500, Sigma Alrich, St. Louis, Missouri, USA).