Sc 390806

Antigen retrieval of all paraffin-embedded kidney sections was performed before immuno-staining by microwave heating method in EDTA (MVS-0098, MXB Biotechnologies, Foochow, China) and Citrate antigen retrieval solution (MVS-0100, MXB Biotechnologies, Foochow, China) for kidney sections from mice and patients, respectively. For IHC, formalin-fixed and paraffin-embedded kidney sections were incubated with primary antibodies against F4/80 (ab6640, Abcam, UK) and CD68 (ab201340, Abcam, UK), an ultrasensitive streptavidin peroxidase detection system was used for detection (MXB Biotechnologies, Foochow, China). Quantitative analysis of the number of positive cells was performed under original magnification (×200) in 20 randomly selected fields per mouse in a blinded fashion. Immunofluorescence staining of paraformaldehyde-fixed kidney sections was performed with primary antibody against Mincle (CLEC-4E (B-7): sc-390806, Santa Cruz, USA), SAP130 (sc-398670, Santa Cruz, USA), and CD68 (ab125212, Abcam, UK), followed by incubation with secondary antibodies. Cell nuclei were stained with DAPI. Immunostained samples were visualized under a confocal microscope (FV3000, Olympus).

The total protein was extracted from cell and tissues using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Proteins (25 μg of each sample) were separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane by wet blotting. After blocking in 5% milk, the membranes were incubated with the antibodies against p-p65 (Rabbit anti-mouse, sc-136548, 1:1000, Santa Cruz), p65 (Mouse anti-mouse, #6956, 1:1000, CST), Mincle (Mouse anti-mouse, sc-390806, 1:500, Santa Cruz), iNOS (Rabbit anti-mouse, 13120S, 1:1000, CST), ZO-1 (Rabbit anti-mouse, 61–7300, 1:1000, Life technologies), Occludin (Mouse anti-mouse, 33–1500, 1:1000, Life technologies), Claudin-1 (Rabbit anti-mouse, 71–7800, 1:1000, Life technologies) and GAPDH (Mouse anti-mouse, AB2000, 1:5000, Abiways) and β-actin (Rabbit anti-mouse, 1:50000, abcam) at 4°C overnight. The next day, membranes were subsequently incubated with the secondary antibody of peroxidase-conjugated goat anti-mouse IgG (ZB-2305, 1:3000) and goat anti-rabbit IgG (ZB-2301, 1:3000) for 1 h at room temperature. The band was analyzed by the gel imaging system. Gray intensity of the band was calculated by ImageJ software.