Rabbit anti mouse c3

Urine protein was measured by a BCA protein assay kit. Urine creatinine was measured using a commercial kit (BioSino Bio-technology and Science Inc., Beijing, China). The data were expressed as milligrams of albumin per millimole creatinine.
For histopathology, kidneys were harvested from exsanguinated mice, immediately fixed in 4% buffered paraformaldehyde, and then embedded in paraffin. Renal sections (4 μm) stained with haematoxylin & eosin (H&E) and periodic acid-Schiff (PAS) were used for morphometric analysis of activity indices for lupus nephritis (LN). For immunofluorescence, tissues were snap-frozen in liquid nitrogen, sectioned and fixed in paraformaldehyde. The sections were stained with Texas Red 594-labelled donkey anti-goat IgG (1:100; Invitrogen, USA) at room temperature or rabbit anti-mouse C3 (1:100; Abcam, Cambridge, UK) and then incubated with Texas Red 594-labelled donkey anti-rabbit IgG (1:100; Invitrogen, USA). Nuclei were stained with DAPI (1:1,000; Invitrogen, USA). Sections were visualized with a Nikon Eclipse Ti-U fluorescence microscope (Nikon, Japan). The fluorescence intensity of the glomeruli from each animal was calculated using Image-Pro Plus v6.0.

Slides were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 min and measured with 0.2% Triton-X 100 for 10 min. Then, they were blocked with 5% bovine serum albumin and incubated with Alexa Fluor 555-antimouse IgG (H + L) (Cell Signaling Technology, MA, USA) for 1 hr at room temperature. For the staining of complement 3 (C3), frozen sections were incubated with rabbit antimouse C3 (Abcam, MA, USA) followed by the staining of secondary Alexa Fluor 488-antirabbit IgG (H + L) (Proteintech, Wuhan, China). After three PBS washes, the slides were counterstained with DAPI for 1 min and then observed under the Confocal Laser Scanning Microscope FV3000 (Olympus, Tokyo, Japan).

Frozen sections were fixed in 100% acetone and 1% paraformaldehyde, stained with rabbit anti-mouse IgG (Abcam) and rabbit anti-mouse C3 (Abcam) and then stained with goat anti-rabbit IgG (Abcam). The sections were observed with a fluorescence microscope (ZEISS, Germany), and the MFI of glomeruli in different groups was calculated using ImageJ software (National Institutes of Health, USA).