Fitc conjugated anti rabbit igg

The cells were fixed in 4% paraformaldehyde for 20 min and incubated in Triton-X100 for 10 min to penetrate the cell membrane. Next, a goat serum blocking solution was added to each well for 1 h. Next, the cells were incubated with rabbit anti-Nrf2 antibody (Abcam, Cambridge, MA, USA; 1:100) overnight at 4 °C and further incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Abcam, Cambridge, MA, USA; 1:1000) for 1 h at room temperature in the dark, and images were captured under a fluorescence microscope (LEICA, Wetzlar, Germany).

A 48‐well culture plate was coated with 300 μg/ml of acid‐soluble type I collagen from the bovine tendon (Nano Zist Arayeh, Iran), as described in previous methods.²⁷ The plate was blocked with 1% BSA in PBS‐T (PBS and 0.1% Tween 20) for 2 h at 37°C. In the next step, KGF and vibrioCBD‐KGF at 50 ng/ml concentration were added into separate wells and incubated at 37°C for 2 h. After extensive washing with PBS‐T, rabbit anti‐KGF antibody (Sigma, USA) was applied at 1:1000 dilution to the plate as the primary antibody. The plate was incubated overnight at 4°C and washed three times with PBS‐T. FITC‐conjugated anti‐rabbit IgG (1:1000, Abcam, UK) was added to the wells as the secondary antibody. Subsequently, the plate was incubated at 37°C for an hour, followed by three washes with PBS‐T. The interaction of proteins with collagen was visualized in the 48‐well plate under fluorescence microscopy (Labomed, USA). Scanning Electron Microscopy (SEM) and Picro‐sirius red staining were used to confirm that collagen was coated and remained on the surface of wells after washing processes.^[28, ^29]

The human OA chondrocytes were seeded on the cover slips in 6-well plates and then incubated for 24 h with or without NBP (0.1 μM). Next, the cells were rinsed in PBS and then fixed with 4% paraformaldehyde. Subsequently, the cells were permeabilized with 0.1% Triton X-100 and blocked with 5% bovine serum albumin. Furthermore, the cells were incubated with the primary antibodies against type II collagen (COLII, Abcam, 1 : 200), ACAN (Bioss, 1 : 100), and FoxO3a at 4°C overnight. Afterward, the cells were incubated with FITC conjugated anti-rabbit IgG (Abcam, 1 : 1000) or anti-mouse IgG (Abcam, 1 : 1000) for 1 h in the dark. The nuclei were stained with DAPI (Beyotime, China). Lastly, the cover slips were observed under a fluorescence microscope.

Cultured chondrocytes were rinsed in PBS, and then fixed with 10% neutral buffered formalin for 30 min at room temperature. Triton X-100 (Beyotime Biotechnology) was used to penetrate the cell membrane for 5 min, and donkey serum (Beyotime Biotechnology) was applied to block nonspecific binding sites for 1 h. Cultured cells were incubated with primary antibodies against type II collagen (Abcam; 1:200) and SOD3 (Santa Cruz Biotechnology; 1:100) at 4 °C overnight. The cells were then washed with PBS three times, and then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Abcam; 1:1000) or anti-mouse IgG (Biolegend, USA; 1:200) for 1 h. Nuclei were stained with Hoechst 33258 for 5 min. Finally, the samples were rinsed with PBS and visualized using confocal microscopy. LAS_X software (Flexera software LLC) was applied to quantify the fluorescence intensity of rat chondrocytes from at least 6 images.

Cultured PVNS FLS were fixed in 4% paraformaldehyde for 15 min and incubated in Triton-X100 (Beyotime Biotechnology) for 10 min to penetrate the cell membrane. Next, a goat serum blocking solution (Beyotime Biotechnology) was added to each well for 1 h. Next, the cells were incubated with rabbit anti-cadherin-11 (Abcam, Cambridge, MA, USA; 1:100) overnight at 4°C and further incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Abcam, Cambridge, MA, USA; 1:1000) for 1 h at room temperature in the dark. Finally, the samples were counterstained with phalloidin (Sigma, USA; 1:500) and DAPI (Sigma, USA; 1:1000) for 10 min, and images were captured under a fluorescence microscope (LEICA, USA).

Endometrial stromal cells (ESCs) were isolated from uterine horns of 8- to 10-week-old female SD rats in the estrus phase according to previously described methods (11 (link)). Briefly, the tissue fragments were incubated in a 1:1 mixture of 0.25% Trypsin and 1 mg/ml dispase (Gibco, USA) medium for 60 min at 4°C, followed by digestion for 45 min at 23°C, and 15 min at 37°C. Trypsin activity was inhibited by adding ESC medium [DMEM/F12 containing 10% FBS and 1% penicillin–streptomycin (PS), Gibco, USA]. After rinsing three times with Hanks’ Balanced Salt Solution (HBBS), samples were subject to digestion with 1 mg/ml collagenase at 37°C for 30 min. Stromal cells were collected by passing the cell suspensions through a 40-μm nylon mesh, followed by centrifugation at 500 g for 7 min. Cell pellet was resuspended in ESC medium and plated at the desired density for passaging.
Immunocytochemical staining was performed to characterize ESCs by incubating cells with monoclonal mouse anti-vimentin antibody (1:1,000, ab8978, abcam) and monoclonal rabbit anti-Cytokeratin18 antibody (1:300, ab133263, abcam), respectively. TRITC-conjugated anti-mouse IgG (goat, 1:50, abcam) and FITC-conjugated anti-rabbit IgG (goat, 1:50, abcam) were used as secondary antibody as indicated.