Biotinylated goat anti rabbit igg

The sections were washed three times by 0.01 M PBS and treated with 0.3% hydrogen peroxide methanol solution for 20 min to suppress endogenous peroxidases activity. After washing thrice in PBS, these sections were treated for 30 min at room temperature with 5% bovine serum albumin for the purpose of reducing nonspecific binding and then incubated overnight with rabbit anti-rat TH (1 : 1,000, GeneTex) [27 (link)] and rabbit anti-rat CP (1 : 1,000, Epitomics) [28 (link)] at 4°C in a humidified chamber. The sections were washed with PBS and incubated with biotinylated goat anti-rabbit IgG (Boster) for 30 min at 37°C, accompanied with enlargement through streptavidin peroxidase for 30 min at the same temperature. Afterwards, sections were rinsed by PBS and coloured with DAB kits for 10 min.

Sections of cartilage nodule tissue were deparaffinized and rehydrated and then treated with 3% H₂O₂. Slides were next incubated in 0.01 M citrate buffer for 20 min at 94–98°C and blocked with 5% BSA. Primary antibodies included rabbit anti-human collagen type II antibodies (1:80, Sigma-Aldrich). The staining was visualized using a microscope (Axioskop 40, Zeiss) by applying streptavidin-biotin complex reagent (Boster) and 3,3′-diaminobenzidine (DAB; Boster) after the treatment with a solution of biotinylated goat anti-rabbit IgG (Boster). Sections were treated using the same process but without incubation with a primary antibody as the negative control.

Liver pathological alterations were presented by the established method of our lab (Li et al., 2021 (link)). In brief, liver tissues were fixed, then dehydrated and embedded in paraffin. Paraffin-embedded tissue was cut into 4 μm sections and stained with hematoxylin-eosin (H&E) according to the standard process (Kohypath, Shanghai, China). For Oil Red O (ORO) staining, frozen liver tissues were embedded in Tissue-Tek OCT Compound (Sakura, Tokyo, Japan), cut into ∼8 μm sections, and stained with ORO reagent (Sigma, St. Louis, MO, United States). For immunohistochemical (IHC) analysis, anti-F4/80 (70076 s, cell signaling technology) primary antibody, and biotinylated goat anti-rabbit IgG (BOSTER, SA1022) were applied. Images were captured under a Nikon Eclipse 50i microscope (Nikon, Tokyo, Japan) with a magnification of ×200.

Formalin‐fixed, paraffin‐embedded specimens (5 μm) from the samples were used for immunohistochemical analysis. The slides were incubated with 0.3% H₂O₂ in absolute methanol for 30 min to block endogenous peroxidase activity. The slides were then blocked with phosphate‐buffered saline (PBS)/0.5% bovine serum albumin (BSA) for 30 min at room temperature and overnight incubation with primary antibody against PRR (rabbit polyclonal immunoglobulin G (IgG); Abcam) at 4℃. This was followed by incubation with a biotinylated goat antirabbit IgG diluted 1: 200 in PBS as a secondary antibody (Boster Biological Technology Co., Ltd) in a humidified box at room temperature for 60 min. The slides were then incubated with 50 ml of diaminobenzadine (Boster Biological Technology Co., Ltd) as a substrate, and counterstained with 10% Mayer's hematoxylin (Leagene) and cover‐slipped with Permount (HAORAN Biological Technology Co., Ltd), mounted and observed with a Leica fluorescence microscope (Leica Microsystems). A semi‐quantitative method was used to evaluate the percentage of positive staining area in the glomeruli. Images were analyzed with Image‐Pro Plus 4.5 software (Media Cybernetics, Inc.). The brown areas were judged as positive.