Ab6586

Organoids and PDTX were Hematoxilin and Eosin stained. Immunoflourescence staining with COL-IV and ZO-1 antibodies was performed using the anti-Collagen IV rabbit polyclonal (Abcam ref ab6586) and the anti-ZO1 tight junction protein monoclonal antibody (ThermoFisher ref Z01-1A12) antibodies following standard methods.

The primary antibodies against fibronectin (FN, ab2413), type IV collagen (Col-IV, ab6586), quinolinate phosphoribosyltransferase (QPRT, ab171944, for cell Western blotting), nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1, ab45652, for kidney tissue Western blotting and immunofluorescence staining) and nicotinamide riboside kinase 1 (NRK1, ab169548, for cell Western blotting) were purchased from Abcam (Cambridge, MA, USA). Antibodies of anti-vimentin (#5741), anti-α-Tubulin (#3873) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (#7074) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against α-smooth muscle actin (α-SMA, A5228), β-actin (A5441) and QPRT (SAB1410425, for kidney tissue Western blotting and immunofluorescence staining) were from Sigma-Aldrich (St Louis, MO, USA). The anti-NMNAT1 (sc-271557, for cell Western blotting) and anti-NRK1 (sc-398852, for kidney tissue Western blotting and immunofluorescence staining) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against nicotinic acid phosphoribosyltransferase 1 (NAPRT1, 13549-1-AP), nicotinamide phosphoribosyltransferase (NAMPT, 11776-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 60004-1-IG) and HRP-conjugated anti-mouse IgG (SA00001-1) were purchased from Proteintech Group (Wuhan, China).

Transwell inserts containing hfRPE cells were rinsed in PBS, fixed for 10 min in 4% paraformaldehyde (PFA) in PBS followed by fixation in 1% glutaraldehyde for 30 min at room temperature. Inserts were cut into small pieces and blocked with 1% BSA for 30 min at RT. Primary antibodies were incubated overnight at 4 °C. Primary antibodies used were: Col IV (AB6586, Abcam, Cambridge, MA), Col VI (AB6588), Col I (AB34710), FN (AB2413), EFEMP1 (SC33722, Santa Cruz Biotechnology, Santa Cruz, CA), and C3aR (HM2195, Hycult Biotech, Plymouth Meeting, PA). Secondary antibodies labeled with Alexa-488 or Alexa-555 (Life Technologies, Grand Island, NY) were incubated for 1 h at RT. Controls were incubated only with secondary antibody. Sections were mounted with fluoromount G (Electron Microscopy Sciences, Hatfield, PA) and visualized by TCS SP5 II confocal laser scanning microscope (Leica). 90° projections: z-stack was built from images taken every 0.5 µm. Tridimensional 90° projections were performed with ImageJ^{68 (link)}. Quantification of fluorescent signal was performed by converting z-stacks to 8-bit binary images and measuring the integrated intensity with ImageJ^{68 (link)}.