Biotin blocker system

Staining for CD68 and F4/80 was performed on acetone-fixed frozen sections (7 µm) and endogenous biotin was blocked using a biotin blocker system (DAKO, Carpinteria, CA). For TLR4 and WT1 detection, formalin-fixed sections were deparaffinized and boiled in 10 mM sodium citrate buffer (pH 6.0). Sections were then incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies: a rat anti-mouse CD68 antibody (ABD Serotec Inc., Oxford, UK), a rat anti-mouse F4/80 (ABD Serotec Inc.), rabbit anti-mouse-TLR4 antibody (Invitrogen, Catalogue No. 48-2300) for glomerular [22] (link), goat anti-mouse-TLR4 antibody (Santa Cruz Biotechnology, Catalogue No sc-12511) for tubular [8] (link), rabbit anti-WT1 antibody (Abcam, Cambridge, UK), or concentration-matched IgG as an isotype negative control. The sections were exposed to H₂O₂ and then incubated with biotinylated anti-rat IgG or anti-rabbit IgG (BD Biosciences, Pharmingen), or anti-goat IgG (Vector Laboratories Inc). A Vector stain ABC kit (Vector Laboratories Inc) was applied to the tissue followed by DAB solution (DAKO). Immunostaining for α-SMA was performed on formalin-fixed paraffin sections using Dako ARK Peroxidase for Mouse Primary Antibodies (DAKO) according to the manufacturer's instructions.

Staining for WT1 and CD68 were performed on acetone-fixed frozen sections (7μm) after endogenous biotin was blocked using a biotin blocker system (DAKO, Carpinteria, CA). To detect Type 1 Collagen (Col-1), formalin-fixed sections (5μm) were deparaffinised and antigen retrieval performed by boiling sections for 10 minutes in 10 mM sodium citrate buffer (pH 6.0). Sections were then incubated with 10% normal horse serum followed by 60 minute incubation with primary antibodies: rat anti-mouse CD68 (ABD Serotec Inc., Oxford, UK), rabbit anti-WT1 (Abcam, Cambridge, UK), rabbit anti-Col-1 (Abcam, Cambridge, UK) or concentration-matched isotype negative control. Endogenous peroxidase activity in the sections was quenched with H₂O₂ prior to application of biotinylated anti-rat IgG or anti-rabbit IgG (BD Biosciences, Pharmingen). A Vector stain ABC kit (Vector Laboratories Inc) was applied to the tissue followed by DAB solution (DAKO).

Acetone-fixed frozen sections of kidney and spleen were stained for CD4, CD8, CD68, CD11c, and Ly-6B.2 as described previously (Wu et al., 2020 ). Endogenous peroxidase activity was blocked with 0.06% hydrogen peroxide, followed by application of a biotin blocker system (DAKO). After blocking with 20% normal horse serum (NHS), primary antibodies: anti-CD4 (BD, 550280), anti-CD8 (BD, 550281), anti-CD11c (BD, 550283), anti-CD68 (ABD Serotec Inc., MCA1957), and anti-Ly-6B.2 (ABD Serotec Inc., MCA771GA) were incubated for 60 min. For visualization of bound primary antibodies, sections were washed then incubated with secondary antibodies: biotinylated anti-rat, anti-hamster, and anti-rabbit IgG (BD Pharmingen). Vector stain ABC kit (Vector Laboratories Inc.) was applied, followed by 3,3diaminobenzidine (DAB) solution (DAKO), and counter-staining with Harris’ hematoxylin.
Immunohistochemistry staining was assessed in a blinded manner by analysis of 20 consecutive high-power fields (×400 magnification) of renal cortex in each section. Macrophage (CD68⁺) and dendritic cell (CD11c⁺) infiltrates were quantified using digital image analysis software (Image Pro Premier 9). T cells (CD4⁺ or CD8⁺) and neutrophils (Ly-6B.2⁺) were counted using an ocular grid and expressed as cells per HPF in a blinded manner as described previously (Wu et al., 2007 (link), 2020 ).