Cy3 conjugated anti rabbit igg

Twenty-eight specified pathogen-free, 3-month-old, male Sprague Dawley (SD) rats, were used for this study. Each rat was housed in our animal facility under pathogen-free conditions and fed a standard laboratory diet with free access to water. The temperature was maintained at 18–22°C with a 12-h light/dark cycle. All animal procedures complied with government-published recommendations for the use of laboratory animals. The study was approved by the Institutional Ethics Review Boards of Guangdong Provincial Hospital of Chinese Medicine.
The anti-collagen-I, occludin, 8-hydroxy-2'-deoxyguanosine (8-OHdG), and NF-κB antibodies were procured from Abcam (Cambridge, UK), anti-β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). FITC-conjugated anti-mouse IgG, cy3-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG, and rabbit IgG were purchased from Beyotime Biotechnology Company (Shanghai, China).
Rhubarb granules were purchased from Jiang Yin Tianjiang Pharmaceutical Company (Product Lot: 1303196) and were monitored for contaminants (heavy metals, pesticides, and mycotoxins) before formulation.

Tissues from the rat aortae were fixed overnight in 4% PFA at 4 °C, embedded in OCT compound (Tissue-Tek) and cryosectioned for staining. Sections were air-dried for 2 h, washed 3 times in PBS, permeabilized in 0.5% Triton-X, and blocked for 15 min with 1% BSA in PBS. Fixed tissues were incubated at 4 °C overnight with primary antibodies against p21 (1:100, Abcam) and p16 (1:200, Abcam), and then incubated with secondary antibodies (Cy3-conjugated anti-rabbit IgG, 1:200, Beyotime, Beijing, China) at room temperature for 2 h. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Abcam, Cambridge, MA). Images were captured using a fluorescence microscope (Olympus, Tokyo, Japan).

PTCs were seeded into wells and cultured for 24 h until the cells reached confluence. After stimulation, the cells were fixed in 4% paraformaldehyde for 15 min at 4°C; permeabilized with 0.1% Triton X-100; blocked with 5% bovine serum albumin for 15 min at room temperature; and incubated simultaneously with anti-E-cadherin (1:100) and anti-α-SMA (1:50) at 4°C overnight. Then, the cells were probed with fluorescein isothiocyanate-conjugated anti-mouse IgG (1:100; Beyotime Institute of Biotechnology) and CY3-conjugated anti-rabbit IgG (1:400; Beyotime Institute of Biotechnology) for 1.5 h in dark. Finally, the cells were incubated with Mounting Medium with DAPI (Zhongshan Golden bridge Biotechnology, Beijing, China) in the dark for 5 min. The immunoassayed proteins were observed using a fluorescence microscope (FV10-ASW; Olympus), and the cells were photographed using a confocal microscope (Radiance 2000; Bio-Rad). Renal tissues were embedded in OCT compound. Cryostat sections (4 μm) were fixed in 4% paraformaldehyde and blocked with casein, and then stained directly with anti-E-cadherin and anti-α-SMA. The subsequent procedures were the same as described above.