Anti tsp 1

Glomeruli were isolated from mice by gradually sieving method as previously described [33 (link)]. Briefly, kidneys were minced and digested in collagenase A (Sigma) at 37°C for 30 minutes with gentle agitation. The digested kidney tissues were gently pressed through a 100-μm cell strainer. The filtered tissues were then passed through 70 μm and 40 μm cell strainer. The final filtered tissues were collected and centrifuged at 2,000 rpm for 5 minutes. The pellet enriched in glomeruli was collected. Mouse glomeruli and podocytes were homogenized. Equal amount of total protein was subjected to SDS-PAGE gel under reducing conditions and transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated with anti-TSP1 (Thermo Scientific), anti-CD36 (Novua), anti-phospho-p38 and anti-total p38 antibodies (Cell Signaling) and then with horseradish peroxidase-conjugated secondary antibody (Jackson Labs). The reaction was visualized using an enhanced chemiluminescence system (Pierce). Immunoblots were analyzed by scanning densitometry and quantified by Quantity One gel Analysis software (Bio-Rad Laboratories).

Confluent ECs on glass coated with 1% gelatin were fixed with 4% PFA during 8 min at RT. The cells were stained with anti‐MT1‐MMP (LEM‐2/15) and anti‐TSP1 (Thermo Fisher, MA5‐13398) primary antibodies diluted 1:100. Cells were incubated with appropriate secondary fluorescent antibodies (anti‐mouse IgG, anti‐rabbit IgG from Jackson ImmunoResearch), phalloidin FITC (Life Technologies, F432), and Hoechst 33342 (Invitrogen, H1399) for 30 min at 37°C. Fluorescence microscopy images were acquired with a Zeiss LSM 700 confocal microscope at 21–23°C using Plan‐Apochromat, 40 × 0.8 oil DIC M27, and z‐stacks were captured every 1 μm. Images were analyzed using ImageJ software (https://imagej.nih.gov/ij/).