Collagen 1 antibody

Heart tissues were fixed with 4% paraformaldehyde overnight and then embedded in paraffin. Expressions of nitrotyrosine, TNF-α, TGF-β1, and collagen I were examined on the tissue section using immunohistochemical (IHC) analyses as described previously (Tian et al., 2009 (link)). The dilution concentration of nitrotyrosine antibody (Millipore, Burlington, MA, United States), tumor necrosis factor α (TNF-α) antibody (Abcam, Cambs, United Kingdom), transforming growth factor β1 (TGF-β1) antibody (Abcam, Cambs, United Kingdom), collagen I antibody (Abcam, Cambs, United Kingdom), and CD31 (Abcam, Cambs, United Kingdom) was 1:100, 1:50, 1:200, and 1:300, 1:500, respectively. For IHC analysis, each section was captured in at least 10 pictures and all pictures were quantified. The protein expression intensity was assessed by estimating the area of the objects and the medium pixel intensity per object, as the integrated optical density (IOD). IOD to area ratio was used to present the results. All images were acquired and processed in TIFF format, analysis was done using Image Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, United States). Capillary density was assessed by capillaries/myocyte nucleus (C/M) values. C/M values were counted in 5 fields randomly selected from each slice.

Standard procedure for extracting protein in renal tissue was used. BCA protein assay was employed to determine protein concentration. Protein in lysates were loaded equally and separated on SDS-PAGE gel and transferred to PVDF membrane before blocking. After incubation with 5%BSA, the membranes were incubated with primary antibodies and secondary antibodies in sequence. The Jmjd3 antibody, fibronectin antibody, and collagen I antibody were purchased from Abcam corporation. The α-SMA antibody was obtained from Sigma Corporation. The GAPDH antibody and β-actin antibody were purchased from Santa Cruz. ECL reagents were used for detecting chemiluminescence signals.

The paraffin-embedded tissue sections were dried, deparaffinized, and rehydrated. Following a microwave pretreatment in citrate buffer (pH 6.0), the slides were immersed in 3% hydrogen peroxide for 20 min to block the activity of endogenous peroxidase. After extensive washing with PBS, the slides were incubated with γH2AX (1:480; CST, United States), Cleaved Caspase 3 antibody (1:200; CST, United States), Collagen I antibody (1:200; abcam, United Kingdom) or α-SMA antibody (1:100; abcam, United Kingdom) overnight at 4°C. The sections were then incubated with the secondary antibody for 1 h at room temperature, and the slides were developed using the UltraVision Quanto HRP detection kit (Thermo Scientific, United States). Finally, the slides were counterstained using hematoxylin. The slides were read in blindness, and the images were recorded.