3 3 diaminobenzidine (dab)

The protein expression levels of α-SMA, Col I, TGF-β1 and HIF-1α in renal tissues were detected using immunohistochemical staining. Briefly, paraffin-embedded renal specimens were cut into slices. After dewaxing with xylene and dehydrating with a gradient of increasing ethanol solutions (95% ethanol followed by anhydrous ethanol 3 times, 5-10 sec each time), the renal slices were incubated in hydrogen peroxide solution (0.3%, 37˚C, 10 min) to quench the endogenous peroxidase activity, followed by incubation at room temperature for 20 min with 10% goat serum for blocking non-specific binding. Next, the corresponding primary antibody was added and incubated with the renal sections (4˚C, overnight). The primary antibodies used were: α-SMA (1:6,000; cat. no. BM0002; Cell Signaling Technology, Inc.), Col I (1:1,400; cat. no. BA0325; Cell Signaling Technology, Inc.), TGF-β1 (1:6,000; cat. no. ab179695; Cell Signaling Technology, Inc.) and HIF-1α (1:1,000; cat. no. BS-3514; Novus Biologicals, LLC). After incubation at 37˚C for 1 h with the secondary antibody diluted in PBS (1:100; cat. no. WP151228; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd), the renal sections were treated with DAB (Wuhan Boster Biological Technology, Ltd.) to develop the signal. Finally, renal slices were visualized using Olympus Soft Imaging Solutions. The sections were observed using a light microscope (IX51; Olympus Corporation). The integrated optical density (IOD) was used to quantify the positively stained area in Image Pro-Plus. The average value of 5 randomly selected fields of view (magnification, x400) for each renal sample was calculated. The protein levels of α-SMA, Col I, TGF-β1 and HIF-1α in renal tissues are expressed as the ratio of IOD to the positive staining area.

The kidney tissue was fixed in 4% paraformaldehyde solution at room temperature for 24 h, embedded in paraffin and cut into 4-µm-thick sections. The paraffin-embedded kidney tissue was cut into 4-µm-thick slices, adhered to microscope slides, baked at 58°C for 2 h, dewaxed using xylene twice, hydrated with gradient alcohol (100, 95, 90, 80 and 70%), placed into boiled sodium citrate solution for antigen repair and naturally cooled to room temperature (18–30°C). The sections were rinsed with PBS three times, co-incubated with an endogenous peroxidase blocker at room temperature for 10 min, rinsed with PBS three times and co-incubated with non-immunized animal serum (10%; Fuzhou Maixin Biotech Co., Ltd.) at room temperature for 10 min. After removing the serum, the following primary antibodies were added in a dropwise manner: Rabbit anti-LC3 polyclonal antibody (1:200; cat. no. ab48394; Abcam) and rabbit anti-Beclin-1 polyclonal antibody (1:500; cat. no. ab62557; Abcam). Slides were then incubated overnight at 4°C and then rinsed with PBS three times. Biotin-labeled sheep anti-rabbit IgG (ready to use; cat. no. KIT-9710; Fuzhou Maixin Biotech Co., Ltd.) was added and slides were incubated at room temperature for 10 min, after which they were rinsed with PBS thrice, incubated with streptavidin-peroxidase (Fuzhou Maixin Biotech Co., Ltd.) at room temperature for 10 min and rinsed with PBS three times. Then, the sections were treated with DAB (Wuhan Boster Biological Technology Co., Ltd.) for color development at room temperature for 3 min, rinsed with distilled water, dyed with hematoxylin at room temperature for 1 min and rinsed using tap water for blueness. Gradient alcohol was used for dehydration, sections were dried and made transparent using xylene, following which neutral gum was used for sealing. Brown staining indicated positive expression as observed under an optical microscope (magnification, ×400). Image-pro Plus 6.0 software (Media Cybernetics, Inc.) was used for analysis, and the relative protein expression was represented in terms of mean density.