Anti αsma

For each mouse, quantitative analysis of glomerular volume, fractional mesangial area, and tuft area in paraffin-embedded kidney sections stained with PAS reagent was performed as described previously [33 (link)]. To examine collagen matrix, paraffin-embedded sections were stained with a picrosirius red stain [33 (link)]. Oil Red O staining was performed to evaluate lipid accumulation in frozen kidney tissues as described previously [33 (link)]. For immunohistochemistry, we used anti-nephrin (1:100; Progen biotechnik GmbH, Heidelberg, Germany), anti-F4/80 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-αSMA (1:200), and anti-collagen IV (1:200; Southern Biotechnology Associates, Birmingham, AL, USA) antibodies. Images were captured using a Zeiss microscope equipped with an Axio Cam HRC digital camera and Axio Cam software (Carl Zeiss, Thornwood, NY, USA). Staining intensities were then quantified using Image-Pro Plus 4.5 software (Media 149 Cybernetics, Silver Springs, MD, USA) as described previously [33 (link)]. Imaging for DAPI (1:1000), anti-PMP70 (1:200, Abcam, Cambridge, MA), and anti-catalase (1:200, Santa Cruz Biotechnology) antibodies was conducted using a confocal microscope (Carl Zeiss, Gottingen, Germany).

Frozen sections (5 μm thickness) were fixed in acetone and blocked with 1% BSA in PBS. The staining for collagen I, collagen III, and αSMA was performed using FITC-conjugated mouse anti-collagen I, anti-αSMA (SouthernBiotech, Birmingham, AL), and anti-collagen III antibodies (Novus Biologicals, Littleton, CO), respectively. Slides were mounted with Vectashield mounting medium with or without 4′,6′-diamidino-2-phenylindole (Vector Labs, Burlingame, CA) and visualized using an EVOS FL Auto microscope (Life Technologies, Carlsbad, CA).