Rabbit anti c3 antibody

Mouse kidney and spleen tissues were fixed in formalin and embedded in paraffin. H&E staining was used to assess the histological features of the kidney. The renal pathology was scored according to the criteria of previous studies (64 (link)). To assess the immune complex deposition in the kidney, we stained paraffin-embedded renal sections with rabbit anti-C3 antibody (Abcam) and HRP-conjugated anti-rabbit antibody (Abcam) for mouse C3 and HRP-conjugated anti-mouse IgG antibody (Abcam) for mouse IgG. PNA (GC zone), CD3 (T cell zone), and B220 (B cell zone) were used to determine the GC area. For PNA staining, spleen tissues were stained with biotinylated anti-peanut agglutinin (Vector Laboratories ), and then incubated with biotinylated anti-peanut agglutinin antibody (Vector Laboratories). For CD3 staining, rabbit anti-CD3 antibody (Abcam) and HRP-conjugated anti-rabbit antibody (Abcam) were used. For B220 (CD45R) staining, rat anti-mouse CD45R antibody (Abcam) and HRP-conjugated goat anti-rat IgG(H+L) antibody (Proteintech) were used. After incubation of primary antibody and HRP-conjugated antibody, the opal 7-Color Manual IHC Kit (Perkin Elmer) was used for fluorescence labeling. Images were captured by Perkin Elmer, and the images were analyzed by the Mantra system. Information on antibodies is provided in Supplemental Table 2.

The vertebra segments were harvested from 6 experimental models of each time point, post-fixed and sectioned. Sections were allowed to incubate with rabbit anti-C3 antibody (1:200 dilution, Abcam), rabbit anti-S100A10 antibody (1:200 dilution, Abcam), rabbit anti-HSF1 antibody (1:200 dilution, CST, 12972), mouse anti-S100β antibody (1:400 dilution, Sigma), or mouse anti-human GFAP antibody (1:400 dilution, Sigma) at 4 °C for 36 h. The sections were further reacted with the FITC-labeled secondary antibody goat anti-mouse IgG (1:400 dilution, Gibco), or the TRITC-labeled secondary antibody donkey anti-rabbit IgG (1:400 dilution, Gibco) at 4 °C overnight, followed by observation under a confocal laser scanning microscope (Leica, Heidelberg, Germany).

Sections were boiled in citric acid buffer and were treated with 0.3% (v/v) Triton X-100 and 10% (v/v) goat serum, they were incubated overnight at 4 °C with primary antibodies (rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) antibody, Wako, Japan; mouse anti-NLRP3 antibody, Thermo Fisher, USA; mouse purified anti-β-amyloid, 1-42 antibody, BioLegend, USA; mouse purified anti-β-amyloid, 1-40 antibody, BioLegend, USA; rabbit anti-collagen I antibody, Abcam, USA; rat anti-LAMP2 antibody, Abcam, USA; mouse anti-GFAP antibody, Sigma, USA; rabbit anti-C3 antibody, Abcam, USA; rabbit anti-NeuN antibody, Millipore, USA; mouse anti-cleaved caspase-3 antibody, Cell Signaling Technology, USA) and then incubated with secondary antibodies. Apoptosis was detected using a transferase-mediated deoxyuridine triphosphate-biotin nick end labeling Kit (TUNEL Apoptosis Detection Kit, Roche, Switzerland). Slices were embedded using Fluoroshield^™ with DAPI (Sigma, USA). Images were acquired using a Nikon fluorescence microscope (Nikon, Japan) or a confocal microscope (Leica, Germany).