Tnf α

Paraffin embedded myocardial samples were dewaxed and rehydrated in xylene and ethyl alcohol followed by incubation in 0.3% methanol/H₂O₂ to block endogenous peroxidases. After antigen retrieval was performed in citrate buffer (pH 6.0, 95–100 °C), the sections were incubated overnight with the following primary antibodies (α-SMA,CD31, 1:200, Cell Signaling Technology, USA; TGF-β, TNF-α, IL-1β, Abcam, USA) at 4 °C. Two-step technique (SuperPictureTM3rd Gen IHC Detection kit; Invitrogen, CA, USA) was used for visualization, using 3, 3′-diaminobenzidine (DAB, 0.1 mg/ml, 0.02% H₂O₂, Vector Laboratories, Burlingame, USA) as the chromogen. Sections were counterstained with hematoxylin. We selected the same slide which contained the largest infarct size for each group to ensure the comparability of the three groups. For each slide, 5 separate fields were examined randomly and digitized by microscopy at a magnification of 400×. All five fields were in the same position in all three groups. Image-Pro Plus software (Media Cybernetics, Rockville, USA) was used to determine the area of α-SMA, CD31, TGF-β, TNF-α and IL-1β positive staining.

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.

The cartilage samples were dissected along the axial plane into pieces of 10 mm ∗ 5 mm ∗ 7 mm with a thin layer of subchondral bone and were fixed in 4% formaldehyde for over 24 hours in room temperature. After being decalcified in 10% ethylene diamine tetraacetic acid (EDTA) solution for over 2 weeks, the samples were embedded in paraffin. The cartilage specimens were cut longitudinally into 4 μm thick sections and stained with haematoxylin-eosin (H&E) and safranin-O. We used the Mankin score [33 (link)] (Table 1) to evaluate the cartilage degradation.
Immunohistochemistry was also performed. We use primary antibodies for ADAMTS-7 (1 : 200; Abcam Biotechnology Co., Ltd., London, UK), TNF-α (1 : 200, Beyotime Institute of Biotechnology, Shanghai, China), and Phospho-NF-κB P65 (1 : 100, Bioss Institute of Biotechnology, Beijing, China). A goat anti-rabbit immunoglobulin- (IgG-) horseradish peroxidase (HRP) secondary antibody (1 : 200; Beijing Golden Bridge Biotechnology Co., Ltd., Beijing, China) was applied. Images were captured by a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). Image-Pro Plus software (Media Cybernetics) was used to qualify the average optical density of the ADAMTS-7 positive, TNF-α positive, and Phospho-NF-κB P65 positive areas at 400x magnification.