Biotin conjugated secondary igg

After deparaffinization, the sections were incubation with rabbit anti-8-hydroxydeoxyguanosine (8-OHdG) (diluted 1:500; Abcam, Cambridge, MA, USA), with biotin-conjugated secondary IgG (diluted 1:200; Vector Laboratories, Burlingame, CA, USA), with the avidin-biotin-peroxidase complex (ABC Elite kit, Vector Laboratories), and with diaminobenzidine tetrahydrochloride. The sections were visualized under light microscopy, and digital images were captured and analyzed.

After deparaffinization, the sections were incubated with primary antibodies against polyclonal anti-AQP5 (Abcam, Cambridge, UK) followed by biotin-conjugated secondary IgG (diluted 1:200; Vector Laboratories, Burlingame, CA, USA), avidin–biotin–peroxidase complex (ABC Elite Kit; Vector Laboratories), and diaminobenzidine tetrahydrochloride. Next, we visualized the sections by light microscopy and captured and analyzed digital images. Data was analyzed by signal intensity using NIS-Elements BR 3.2 (Nikon, Japan) in 10 random fields and described as fold change. The fold changes are calculated as the ratio of the final value in each group to the value in control group at day 30 (set as “1”).

After deparaffinization, the sections were incubated with primary antibodies to polyclonal anti-Hif-1a (diluted 1:100; ab2185, Abcam), monoclonal anti-GLUT1 (diluted 1:200; ab40084, Abcam), monoclonal anti-Ki67 (diluted 1:50; ab16667, Abcam), monoclonal anti-PCNA (diluted 1:200; sc-56, Santa Cruz Biotechnology, Dallas, TX, USA), and monoclonal anti-8-hydroxydeoxyguanosine (diluted 1:500; ab62623, Abcam); with biotin-conjugated secondary IgG (diluted 1:200; Vector Laboratories, Burlingame, CA, USA); with the avidin-biotin-peroxidase complex (ABC Elite Kit, Vector Laboratories); and with diaminobenzidine tetrahydrochloride. We then visualized the sections under light microscopy and captured and analyzed the digital images.

After deparaffinization, sections were incubated with primary antibodies against polyclonal anti-light chain 3B (LC3B; Cell Signaling Technology, Danvers, MA, USA), beta-galactosidase (β-gal), and 8-hydroxydeoxyguanosine (8-OHdG) (both from Abcam, Cambridge, MA, USA). Then, biotin-conjugated secondary IgG (1 : 200 dilution; Vector Laboratories, Burlingame, CA, USA), an avidin-biotin-peroxidase complex (Elite ABC Kit; Vector Laboratories), and DAB were added. We visualized sections under a light microscope and captured and analyzed digital images.

After deparaffinization, sections were sequentially treated with 1% H₂O₂ for 10 min and rinsed thoroughly with PBS. Sections were blocked with 2% normal goat serum in PBS at room temperature for 60 min to minimize nonspecific IgG binding, followed by incubation with rabbit anti-TGF-ß1 (diluted 1∶50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti–8-OHdG (diluted 1∶500; abcam, Cambridge, MA, USA), and – mouse anti–MDA (diluted 1∶500; abcam, Cambridge, MA, USA). The samples were washed with PBS, incubated for 60 min at room temperature with biotin-conjugated secondary IgG (diluted 1∶200; Vector Laboratories, Burlingame, CA, USA), diluted in 2% normal blocking serum, and then incubated for 60 min at room temperature with avidin-biotin-peroxidase complex (ABC Elite kit, Vector Laboratories). The samples were then washed with PBS and incubated for 3 min with diaminobenzidine tetrahydrochloride containing 0.05% hydrogen peroxidase to develop the color. Samples were then stained with Mayer's hematoxylin stain. The sections were visualized under light microscopy and digital images were captured and analyzed.

After deparaffinization, the sections were incubated with monoclonal antibodies against ICAM-1 (BD Bioscience, Franklin Lakes, NJ, USA) or α-SMA (Sigma), or polyclonal antibodies against F4/80 (Santa Cruz Biotechnology) or 8-OHdG (Abcam); they were then incubated with biotin-conjugated secondary IgG (diluted 1:200; Vector Laboratories, Burlingame, CA, USA), avidin-biotin-peroxidase complex (ABC Elite Kit; Vector Laboratories), and DAB. Next, sections were visualized by light microscopy; digital images were captured and analyzed using NIS-Elements BR 3.2. Semiquantitative analysis was performed by counting the number of immunohistochemically stained positive cells per field in the renal tissue at ×400 magnification.