Sc 14939

Cultured cells or cryosections of kidney samples were fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized, and stained with primary antibodies against zonula occludens (ZO)-1 (617300, Invitrogen, MD, USA), E-Cadherin (3195, Cell Signaling Technology), vimentin (sc-6260, Santa Cruz Biotechnology), fibronectin (ab2413, Abcam, San Francisco, CA, USA), pH3, and CTGF (sc-14939, Santa Cruz Biotechnology), followed by Alexa Fluor–conjugated secondary antibody staining (Life Technologies, Carlsbad, CA, USA). Finally, cells or cryosections were mounted with mounting media containing propidium iodide or 4,6-diamidino-2-phenylindole (DAPI, Abcam), and visualized using the EVOS FL microscope (Thermo Fisher Scientific) or the Cytation 5 cell imaging system (BioTek Instruments, Winooski, VT, USA).

AMOTL1 (HPA001196, Sigma), YAP1 (ab52771, Abcam), CTGF antibody (sc-14939, Santa Cruz), Ki67 (550609, BD Pharmingen), and cleaved-Caspase 3 (#9664, CST) were commercially available. The immunohistochemistry and the scoring of the results were performed as previously described [13 (link)]. For immunocytochemistry studies, AGS was cultured on coverslips in a six-well plate. After removal of the medium and three times of PBS washing, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. Followed by three times of PBS washing, the cells were then permeated with 0.1% Triton X-100 at room temperature for 15 min, and another PBS washing three times. Room-temperature blocking was done with 2% BSA for 45 min; then cells were incubated with the primary antibody (1:200) at 4 °C overnight. Again, after one time of PBS washing, the cells were incubated with goat anti-mouse IgG secondary antibody (Alexa Fluor 594, 1:400, Thermo Fisher Scientific) in the dark at room temperature for 1 h. After washing, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific). Images were captured with a microscope (Carl Zeiss Axio Imager 2, Oberkochen, Germany).

Plasma and peritoneal fluid samples were thawed on the day of use and any solids pelleted by centrifugation. Afterwards, the supernatant was diluted 1∶10 in assay buffer. CCN2 present in the plasma or peritoneal fluid was then quantified using an ELISA as previously reported [41] (link). Briefly, the ELISA plate was first coated with CCN2 L-20 antibody, as a specific trapping antibody (sc-14939, Santa Cruz Biotechnology Inc., Dallas, TX, USA). The plates were incubated with sample or CCN2 standard and afterwards washed before the addition of 20a anti-CCN2 antibody. After further washing, horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit; Jackson ImmunoResearch, West Grove, PA, USA) was added, followed by horseradish peroxidase substrate (Enhanced K-Blue TMB substrate, 308175; Neogen Corp., Lexington, KY, USA). The color intensity was allowed to develop, and read at 650 nm using a microplate reader. All CCN2 levels are expressed as ng/ml.