Anti c3

Kidney tissues were stained with anti-C3 (Abcam, Cambridge, UK) at 4 °C overnight, followed by 2 h incubation with secondary antibodies conjugated to Alexa488. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA). Isotype control staining was conducted via probing with rat/rabbit/mouse IgG, rather than primary antibodies. Confocal images were acquired using an LSM 800 confocal microscope (Zeiss, Oberkochen, Germany).

The following primary antibodies were used in immunocytochemistry and immunohistochemistry studies: anti-C1q (Invitrogen, MA1-40311, lot TD2550204, 1:50); anti-C3 (Abcam, ab200999, lot GR294205-17, 1:200; Hycult, HM1065, lot 23152M1017-A, 1:50); anti-Iba-1 (Abcam, ab178847, lot GR3229566-2, 1:100; GeneTex, GTX101495, lot 41885, 1:50); anti-GFAP (Cell Signaling Technology, 3670S, lot 5, 1:100); anti-LAMP1 (Abcam, ab24170, lot GR3235359-1, 1:100); anti-MAP2 (Abcam, ab11267, lot GR281093-9, 1:100); anti-PSD95 (Abcam, ab12093, lot GR3271883-1; ab18258, lot GR3174013-1, 1:100); anti-Synaptophysin (Abcam, ab32127, lot GR312544-1, 1:100); 4G8 (Biolegend, 800704, lot B238676, 1:100); anti-β-actin (MBL, M177-3, lot 002, 1:1000); mouse anti-rat CD11b (BD Biosciences, 554980, lot 6294728); mouse IgA, κ isotype control (BD Biosciences, 553476, lot 7257918); mouse anti-rat CD32 (BD Biosciences, 550273, lot 8339705); mouse IgG1κ isotype control (BD Biosciences, 553447, lot 8241620).

The cSCC cells or frozen tumour tissues were lysed by RIPA lysis buffer. Proteins were extracted, measured and analysed by western blotting. Briefly, 40 μg per lane of protein was separated by 10% (w/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane. Then, 5% (w/v) bovine serum albumin (BSA) was used to block nonspecific binding in the membrane. After washing with Tris buffered saline with Tween buffer, membranes were incubated overnight at 4°C with primary antibodies. Primary antibodies included anti‐C3 (1:800; Abcam, Cambridge, MA, USA), anti‐cyclin D1 (1:800; Abcam), anti‐cyclin E1 (1:800; Abcam), anti‐VEGF (1:800; Abcam), anti‐pro‐MMP1 (1:800; Abcam), anti‐pro‐MMP2 (1:800; Abcam), anti‐Sox‐2 (1:800; Abcam) and anti‐DKK‐1 (1:800; Abcam). Horseradish‐peroxidase‐conjugated secondary antibody was then added, and cells were incubated for 2 hours at room temperature. Signals of target protein were visualized by incubating the membranes with chemiluminescence reagents and then quantifying with the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Β‐actin was used as the loading control for normalization.

As a marker of neuronal inflammation, microglial activation, astrocyte activation, and astrocytosis were determined by using ionised calcium-binding adaptor molecule 1 (Iba-1), complement component 3 (C3), and GFAP, respectively. For Iba-1 staining, 20 μm cryosections prepared from fixed frozen right hemispheres were incubated in 10 mM T.E. buffer (pH 6.0) for 20 hours at 37°C for antigen retrieval. The sections were then incubated with a primary antibody of anti-Iba-1 (1:500, Novachem), anti-C3 (1:100, Abcam (Cambridge, United Kingdom)), or anti-GFAP (1:200, Abcam) for 20 hours at 4°C. Subsequently, Iba-1 and GFAP were detected by using anti-rabbit IgG conjugated with Alexa488, while C3 was detected with anti-rat IgG Alexa488 for 2 hours at 20°C. Confocal images were randomly captured with UltraVIEW Vox with 20X objective by a blinded investigator. Zeiss ZEN Intellisis trainable segmentation module was used to identify the stained astrocytes and microglia. The intensity of the staining was calculated per image.