Tgf β1

Histological injury was performed in 4-μm methyl Carnoy's-fixed paraffin sections stained with
Periodic acid-Schiff (PAS). Immnunostaining was performed on paraffin sections using a microwave-based antigen retrieval technique [27 (link)]. Primary antibodies used in the study were as followed: collagen I (Southern Technology, Birmingham, AL), α-SMA (Sigma, St. Louis, MO), TNFα, MCP-1, TGF-β1, phospho-Smad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-NFκB/p65, CD3 (Abcam, Cambridge, MA), and F4/80 (Serotec, Oxford, UK). After being immunostained with the secondary antibodies, sections were developed with diaminobenzidine to produce a brown color. All slides were counterstained with hematoxylin except for phospho-Smad2/3 and phospho-NFκB/p65 immunodetection. The percentage of positive staining for collagen I, TNFα, MCP-1, TGF-β1 was measured by using a quantitative image-analysis system (Image-Pro Plus 6.5, Media Cybernetics, Silver Spring, MD) [28 (link), 29 (link)], while the number of positive phopsho-p65, phospho-Smad2/3, CD3, F4/80+ cells in the tubulointerstitium were counted under high-power fields (×40) by means of a 0.0625-mm² graticule fitted in the eyepiece of the microscope and expressed as cells per millimeters squared.

Formalin-fixed, paraffin-embedded kidney specimens were prepared as 3-μm-thick sections. For general histology, sections were processed for Masson trichrome staining according to the manufacturer’s instructions (Solarbio Life Science, Beijing, China). The collagen volume fractions of Masson staining were quantified by the ImageJ program (National Institutes of Health, Bethesda, MD). For immunohistochemistry staining, sections were incubated with primary antibody against TGFβ1 (Santa Cruz Biotechnology, TX, USA), p16^INK4A (Abcam, MA, USA), Serpin E1 (Santa Cruz Biotechnology), followed by incubation with secondary antibodies and the use of diaminobenzidine substrate kit (Servicebio, Shanghai, China). As negative controls, the primary antibodies were replaced by nonimmune serum from the same species and no nonspecific staining was observed. Computerized morphometry of immunohistochemical staining of TGFβ1, p16^INK4A and Serpin E1 was performed as described previously^{18 (link)} by using Image-Pro Plus software (Media Cybernetics).