Podocin antibody

Indirect immunofluorescent staining was used to determine colocalization of the inflammasome proteins in glomeruli of the mouse kidney, which indicate the formation of inflammasome molecular complex. Frozen kidney tissue slides were fixed in acetone and then incubated overnight at 4°C with either goat anti- Nlrp3 (1:100, Novus Biologicals) and rabbit anti-Asc (1:50, Enzo Lifesciences), or goat anti- Nlrp3 (1:200) and mouse anti-caspase-1 (1:100, Santa Cruz Biotechnology). To further confirm the presence of the inflammasomes specifically in podocytes of the mouse glomeruli, Nlrp3 or caspase-1 was co incubated with a podocin antibody (1:400; Sigma, St. Louis, MO). Double immunofluorescent staining was achieved by incubating with either Alexa-488 or Alexa-555-labeled secondary antibodies for 1 hour at room temperature. After washing, slides were mounted with a DAPI-containing mounting solution, and then observed with a confocal laser scanning microscope (Fluoview FV1000, Olympus, Japan). As previously described [17 (link), 40 (link)], images were analyzed by the Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD), where colocalization was measured and expressed as the Pearson Correlation Coefficient (PCC).

Serum samples were analyzed for the levels of angiogenic markers (sFlt-1, PlGF, and endoglin) using Quantikine ELISA (enzyme-linked immunosorbent assay) kits (R&D Systems, Minneapolis, MN). Aliquots of the midstream urine samples (50–100 mL) were collected in sterile containers and processed as previously described [9] . Briefly, urine albumin, total protein, and creatinine concentrations were measured using standard methods on a Hitachi 911 Chemistry Analyzer (Roche Diagnostics, Indianapolis, IN) and a predicted 24-hour urine protein was estimated from the protein/creatinine (P/Cr) ratio. Urinary sediments from the respective specimens were used for podocyte studies. Podocytes were identified by staining with a podocin antibody (1∶200, Sigma) after culturing the urinary sediments for 24 hours as previously described [9] . Nucleated, positive-staining cells were determined to be podocytes (Figure 1).