Anti podocin antibody

Mouse kidney tissues were frozen in OCT (Thermo Fisher Scientific) and sectioned to 5 μm thickness. The slices were subjected to fixation in prechilled acetone for 600 s under -20°C, blocked in 5% Bovine Serum Albumin (BSA) under RT for 60 min, and incubated at 4°C with the primary antibodies below: anti-KL antibody (Santa Cruz, CA, America), anti-Podocin antibody (Abcam, MA, USA), anti-WT-1 antibody (Abcam, MA, America), and anti-Synaptopodin antibody (Santa Cruz, CA, America). Fluorochrome-conjugated secondary antibodies were acquired from Jackson Immune Research Labs. 4′,6-diamidino-2-phenylindole (DAPI) was utilized for nucleus counterstaining.

Immunofluorescence was performed using frozen Sections (10 µm). The following antibodies were used as primary antibodies: monoclonal rat anti-CD31 antibody (BD Biosciences); polyclonal rabbit anti-type IV collagen antibody (Abcam); anti-mouse Alk1 antibody (R&D systems); anti-Nephrin antibody (Abcam); anti-WT1 antibody (Abcam); anti-podocin antibody (Abcam); anti-cleaved-caspase 3 (Cell Signaling); anti-PDGFRB (R&D). Alexa Fluor 488 or 647 conjugated antibodies (ThermoFisher Scientific) were used as secondary reagents and slides were mounted with Fluoroshield/DAPI (Sigma). Images were obtained by confocal microscopy (Olympus Fluoview). For quantification of immunofluorescence, staining intensity and area was quantified using 50 randomly selected glomeruli per kidney section. Brightness and contrast were adjusted on displayed images (identically for compared image sets) and quantified (identical threshold settings for compared image sets) using ImageJ. For patient samples, paraffin-embedded tissues were cut into 4- to 6-μm sections and processed for immunofluorescence. Antigen retrieval was performed in citrate solution pH = 6. The sections were then labeled with anti-human Alk1 antibody (R&D systems). Slides were subsequently exposed to specific AF647-conjugated secondary antibody (ThermoFisher).

Mouse Actn4 cDNA was subcloned into the pmApple and pEGFP vectors. Expression construct of Shp2 were subcloned into pflag-CMV2 vector. Expression construct of mcherry-ACTN1 was obtained from JC Kuo (NYCU). Expression construct of Shp2-SWAP FRET biosensor was provided by Dr. Yingxiao Wang (UCSD) (Sun et al, 2013 (link)). Anti-paxillin antibody and mouse collagen type-IV from BD Biosciences; anti-β-actin, anti-β-tubulin, and anti-flag antibodies, FITC-phalloidin, ROCK inhibitor Y27632, and Shp2 inhibitor IIB-08 from Sigma-Aldrich; FAK inhibitor 14 from Tocris Bioscience, anti-pY397FAK, anti-pY31-paxillin, and anti-ZO-1 antibodies from Invitrogen; anti-pY542 Shp2, anti-pT18/pS19-MLC, and anti-MLC antibodies from Cell Signaling; anti-α-actinin-1 antibody from Chemicon; anti-α-actinin-4 and anti-phosphotyrosine (clone 4G10) antibodies from Millipore; anti-FAK, anti-Shp2, anti-ROCK1, and anti-ROCK2, antibodies from Santa Cruz Biotechnology Inc.; anti-synaptopodin antibody from Novus; anti-podocin antibody from Abcam; λ protein phosphatase from New England BioLabs. Anti-pS1333 ROCK1 and anti-pS1366 ROCK2 antibodies were generated in our laboratory (Chuang et al, 2012 (link), 2013 (link)).