3 3 diaminobenzidine chromogen

Immunohistochemical staining for collagens types I, II and III was performed with the following primary antibodies: mouse monoclonal anti-collagen type I (GTX26308; GenTex, USA; diluted 1:4000), polyclonal rabbit anti-collagen type II (RAH C22; IMTEK, Russia; diluted 1:20) and mouse monoclonal anti-collagen type III (GTX26310; GenTex, USA; diluted 1:8000). Heat-induced antigen retrieval was performed in a citrate buffer (pH 6.0) for 15 min at 97°C in the PT Link Rinse Station (Dako, Denmark). The sections were pretreated with 10 mg/mL hyaluronidase (NPO Microgen, RF) in Tris-buffered saline, pH 7.0 for 1 h at 37°C. Then, secondary goat anti-mouse and goat anti-rabbit antibodies (REAL EnVision; Dako, Denmark) conjugated with horseradish peroxidase were applied and contrasted with 3,3’-diaminobenzidine chromogen (Dako, Denmark). The slides were counterstained with Mayer’s hematoxylin (Sigma-Aldrich, USA). The IHC staining intensity was semi-quantitatively scored from negative (-) to triple-positive (+++) via the independent examination by two professional pathologists.

Formalin-fixed, paraffin-embedded tissue sections (5μm) were deparaffinized in xylene and rehydrated with gradient concentrations of ethanol. Endogenous peroxidase activity was blocked (0.35% H2O2 in PBS buffer), antigens were retrieved by microwaving (350 W), and nonspecific binding was blocked by 1% bovine serum albumin in PBS buffer. Sections were incubated with FOXN3 antibody (1:300) over night. After extensive wash with PBS, the sections were incubated with the HRP-linked secondary antibody (Envision, DakoCytomation) for 1 hour. Slides were then incubated with 3, 3′-diaminobenzidine chromogen (DakoCytomation), counterstained in Meyer's hematoxylin and mounted.

Mice were anesthetized using a zoletil-xylazine cocktail during perfusion with phosphate-buffered saline (PBS). Kidney was vertically dissected and fixed with 10% formalin. Kidney sections (3 μm in thickness) were stained with Periodic acid-Schiff (PAS) stain (Sigma). Kidney pathology was evaluated using a lupus nephritis classification system as described in a previous study (16 (link)). Immunohistochemistry was performed using a Vectastain ABC kit (Vector). Tissue sections were incubated with anti-nephrin (Progen), anti-synaptopodin (Abcam), anti-podocin (Abcam), or isotype control antibodies (Abcam) at 4°C overnight. Staining was developed using 3,3′-diaminobenzidine chromogen (Dako). Sections were counterstained with hematoxylin QS (Vector).

Formalin‐fixed paraffin‐embedded tissue sections were dewaxed, and antigens were retrieved after 10 minutes of boiling in citrate buffer, pH 6 (Dako). Slides were stained with 10 μg/ml of biotinylated DVD antibodies for 1 hour at room temperature and were visualized with streptavidin–HRP complex using 3,3′‐diaminobenzidine chromogen (Dako). Rabbit anti–ICAM‐1 IgG (Abcam) was detected with HRP‐conjugated goat anti‐rabbit IgG (Jackson Immunotools). Mouse anti–von Willebrand factor (Dako) and mouse anti‐CD31 (R&D Systems) were used to identify human vascular endothelial cells, which were revealed with HRP‐conjugated goat anti‐mouse IgG (Santa Cruz Biotechnology). Sections were counterstained with hematoxylin, mounted with Depex mounting medium (Dako), and acquired with a CellSens imaging system (Olympus).

Paraffin-embedded sections were incubated at 4℃ with the following primary monoclonal antibodies: anti–IL-1β, anti–IL-17, anti–IL-6, and anti–TGF-β. The samples were then incubated with the respective secondary biotinylated antibodies, followed by 30 min incubation with a streptavidin–peroxidase complex. The reaction product was developed using 3,3-diaminobenzidine chromogen (Dako, CA, USA).