α sma

After fixation with a stationary liquid, permeabilization with 0.3% Triton X100, and blocking with 5% BSA solution, cultured cells were incubated overnight with the α-SMA (1:200 dilution, ABclonal, Wuhan, China) primary anti-rabbit antibodies at 4° C. After a brief wash with PBS, cells were incubated with fluorescent secondary antibodies for 1 h and then stained with DAPI (1 mg/ml, Beijing Sola Biotechnology Co., Ltd., Beijing, China) for 10 min at room temperature. All antibodies mentioned above were diluted in NCM universal antibody diluent from New Cell and Molecular Biotech (Jiangsu, China). Immunofluorescence was recorded by a fluorescence microscope. The percentage of α-SMA positive cells was calculated by Image-Pro Plus (Media Cybernetics, Rockville, MD, USA).

The left mouse heart tissues were cut, fixed at 4°C with the methyl Carnoy's solution and paraffin‐embedded tissue sections at 4 µm were evaluated by using haematoxylin and eosin (HE) and Masson's trichrome staining, respectively. For immunohistochemistry, microwave‐based antigen retrieval technique was used as previously described.⁶, ¹¹, ¹² After microwaving, the sections were incubated with primary antibodies against tumour necrosis factor α (TNFα), interleukin‐1β (IL‐1β), MCP‐1, and TGF‐β1 (Santa Cruz Biotechnology), collagen I and collagen III (Southern Biotech), α‐SMA (Sigma), p‐Smad3 (600‐401‐919, Rockland) and F4/80 (Serotec Ltd) for overnight at 4°C. After being washed, the sections were incubated with the corresponding secondary antibodies for one hour at room temperature and signals were visualized with diaminobenzidine and counterstained with haematoxylin. Quantitative analysis of p‐Smad3 or F4/80−positive cells was counted at×40 power field under 0.0625 mm,² and the positive counts were expressed as cells per millimetres squared. In addition, the quantitative image analysis system (Image‐Pro Plus 6.5, Media Cybernetics, Silver Spring, MD) was used to analyse the per cent expression of TNFα, IL‐1β, MCP‐1 and TGF‐β, and deposition of collagen I, III and α‐SMA as previously described.⁶, ¹¹, ¹²

Immunoreactivity for α-SMA and CD68 was performed on liver mouse sections. Briefly, 5 µm mouse liver sections were subjected to antigen retrieval at 120 °C for 5 min in a Pascal Pressure Chamber (Dako Cytomation, Glostrup, Denmark) and blocked for 15 min. Then, slides were treated with low-fat milk for 60 min and incubated in a humidified chamber overnight at 4 °C with 1/500 anti-α-SMA (Abcam, Cambridge, UK) or 1/2000 anti-CD68 (Abcam). Subsequently, slides were incubated with a one-step polymer-horseradish peroxidase (EasyPath-Erviegas, São Paulo, Brazil) for 20 min, developed with 3, 3′-diaminobenzidine chromogen (Sigma-Aldrich), and counterstained with Harris hematoxylin. Semi-quantitative analysis was performed using Image-Pro Plus 4.5 software (Media Cybernetics) in 10 randomly selected fields (20× objective for α-SMA and 40× objective for CD68) per section from the left lobe. Data analysis was based on the following calculations [α-SMA⁺ cells area (%) = α-SMA⁺ area/(total field area − vascular luminal area)] and [CD68⁺ density = CD68⁺ cells/total field area (mm²)].

To evaluate the histological damage, collagen-like matrix deposition was stained with Masson's trichrome staining with the ‘Trichrome stain kit’ (Scy Tek Laboratoris, West Logan, UT) according to the manufacturer's instruction. Immunohistochemistry for detection of TGF-β/Smad signaling and renal fibrosis was performed in 4μm paraffin-embedded tissue sections of mouse kidney tissue using a microwave-based antigen retrieval technique [43 (link)]. Primary antibodies used in this study included rabbit polyclonal antibodies to TGF-β1, phosphorylated Smad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA), collagen I (Southern Biotech, Birmingham, AL), α-SMA (Sigma, St. Louis, MO). The number of phospho-Smad2/3 was 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 total collagen-like contents, expression of TGF-β1, and accumulation of collagen I and α-SMA on were measured in Masson's trichrome and immunostained sections by 10 random areas under high-power by using a quantitative image-analysis system (Image-Pro Plus 6.5, Media Cybernetics, Silver Spring, MD) and expressed as the percent positive area examined.

Paraffin-embedded PM sections (4 μm) were incubated with anti-rat α-smooth muscle actin (α-SMA) (Sigma Chemical, St. Louis, USA) and anti-PCNA (DAKO, Glostrup, Denmark). An LSAB-AP System (DAKO) revealed with fast red dye (Sigma), and a NovolinkPolymer Detection System (Leica Microsystems, Newcastle, UK) revealed with diaminobenzidine were employed for antibody detection.
The expressions of α-SMA and PCNA were calculated by the percentage of the positive area relative to the entire field area, using Image-Pro Plus 7.0 software (Media Cybernetics).