Paraffin embedded kidney sections were dewaxed and rehydrated with ethanol, and then stained with H&E to evaluate kidney morphology, and collagen content was determined by Sirius Red staining [25 (link),26 (link)].
For immunohistochemistry staining, antigens were retrieved using citrate buffer (pH 6). Quenching of endogenous peroxidase activity was performed using 3% hydrogen peroxide for 10 min. The kidney sections were blocked with blocking solution for 1 h and then incubated with the primary antibody overnight at 4 °C. The following day, the kidney sections were incubated with the appropriate HRP-conjugated secondary antibody followed by incubation with ABC solution (Vector Laboratories, Newark, NJ, USA). The immunoreactivities were detected by diaminobenzidine (DAB) solution (Vector Laboratories). Finally, the sections were counterstained with hematoxylin. The immunostaining was examined by a microscope equipped with a digital camera [27 (link),28 (link)]. The number of positive cells were qualified using Image Pro-Plus in a blinded manner.
For immunohistochemistry staining, antigens were retrieved using citrate buffer (pH 6). Quenching of endogenous peroxidase activity was performed using 3% hydrogen peroxide for 10 min. The kidney sections were blocked with blocking solution for 1 h and then incubated with the primary antibody overnight at 4 °C. The following day, the kidney sections were incubated with the appropriate HRP-conjugated secondary antibody followed by incubation with ABC solution (Vector Laboratories, Newark, NJ, USA). The immunoreactivities were detected by diaminobenzidine (DAB) solution (Vector Laboratories). Finally, the sections were counterstained with hematoxylin. The immunostaining was examined by a microscope equipped with a digital camera [27 (link),28 (link)]. The number of positive cells were qualified using Image Pro-Plus in a blinded manner.
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