Masson s trichrome stain

One portion of the excised left kidney tissues was embedded in paraffin and 5-µm thick sections were prepared at Mayo Clinic Histology Core facility. The sections were stained with Masson’s trichrome stain using a kit (Thermo Fisher Scientific) to assess the tissue fibrosis. The extent of cell proliferation was assessed using Ki-67 staining, Col-I and Col-IV staining were performed using Rabbit anti human antibodies at 1:1000 dilutions (Rockland Immunochemicals Inc, Limerick, PA) as described else where^{34 ,36 (link)}. Apoptosis was assessed by TUNEL staining using a colorimetric TUNEL staining kit (Trevigen, Gaithersburg, MD) following manufacturer’s protocol except the slides were counterstained with Hematoxylin instead of methyl green. pSMAD3 and pSMAD2 antibodies (Cell Signaling, Danvers, MA) were used at 1:250 dilutions. Images were captured with a Zeiss microscope and the staining intensity was determined using Zen pro-2.3 software (Carl Zeiss AG, Oberkochen, Germany). The Ki-67, TUNEL, pSMAD3 indices were calculated by counting the number of positive cells for either Ki-67, TUNEL, or pSMAD3/total number of cells multiplied by 100. The collagen I and IV indices were calculated by determining the area of cells staining positive for collagen I or IV and dividing by the total area of cells multiplied by 100.