Cardiac Tissue Immunohistochemistry Analysis

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.

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Wu M., Yang Y., Wang M., Zeng F., Li Q., Liu W., Guo S., He M., Wang Y., Huang J., Zhou L., Li Y., Hu J., Gong W, & Zhang Z. (2018). Exogenous Pancreatic Kallikrein Improves Diabetic Cardiomyopathy in Streptozotocin-Induced Diabetes. Frontiers in Pharmacology, 9, 855.

Publication 2018

nitrotyrosine antibody transforming growth factor β1 (TGF-β1) antibody collagen I antibody Image Pro Plus 6.0 software

Antibody Capillaries Collagen i Dilution Embedded paraffin Heart Myocyte Nitrotyrosine Nucleus Paraformaldehyde Protein Ratio Tissue Transforming growth factor β1 Tumor necrosis factor α

Corresponding Organization :

Other organizations : Soochow University, Second Affiliated Hospital of Soochow University, Huashan Hospital, Fudan University, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University

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Variable analysis

independent variables

Nitrotyrosine antibody concentration (1:100)
TNF-α antibody concentration (1:50)
TGF-β1 antibody concentration (1:200)
Collagen I antibody concentration (1:300)
CD31 antibody concentration (1:500)

dependent variables

Nitrotyrosine expression
TNF-α expression
TGF-β1 expression
Collagen I expression
Capillary density (C/M values)

control variables

Heart tissue fixed with 4% paraformaldehyde overnight
Heart tissue embedded in paraffin
Immunohistochemical (IHC) analysis performed as described previously (Tian et al., 2009)
At least 10 pictures captured and quantified per section
Protein expression intensity assessed by integrated optical density (IOD) to area ratio
Images acquired and processed in TIFF format
Image analysis performed using Image Pro Plus 6.0 software

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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