Ab85034

Western blot analysis was performed as described previously^{59 (link)}. Briefly, proteins were electrophoresed in 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The blots were blocked in 5% milk and incubated overnight at 4 °C with the primary antibodies, followed by incubation with anti-rabbit Dye 680CW or anti-mouse Dye 800CW (LI-COR, MO, USA) at 1/10,000 dilution for 1 h. The specific signals and the corresponding band intensities were evaluated using Odyssey Infrared Imaging system (Odyssey, Berlin, Germany). The protein level was normalized against GAPDH. The following antibodies were used in this study: rabbit anti-CSF1 (0.2 µg/ml; Abcam, ab9693), rabbit anti-CX3CL1 (1/5,000 dilution; Abcam, ab85034), and mouse anti-GAPDH (1/10,000 dilution; Bioworld, MB001).

Immunofluorescence staining was performed in kidney tissue embedded in paraffin according to standard pathology protocols. The primary antibodies used were mouse anti-human antibodies CD14 (ab182032, Abcam), CD31 (ab9498, Abcam), CCL2 (MABN712, Millipore), and rabbit anti-human antibodies CD16 (ab203883, Abcam), CD31 (ab32457, Abcam), CX3CL1 (ab85034, Abcam). The slides were placed in a wet chamber followed by the addition of the appropriate primary antibodies at the concentration recommended by the manufacturer (double staining) and incubated overnight at 4°C. The slides were rinsed three times with PBS, and a 1:200 dilution of Alexa Fluor^®594 goat anti-mouse IgG antibody (ab150116, Abcam) and Alexa Fluor^®488 goat anti-rabbit IgG antibody were applied and incubated at 37°C (30 minutes) in the dark. DAPI mounting medium (ab104139, Abcam) was used for nuclear staining. Finally, the slides were viewed under fluorescent microscopy (Olympus BX51; Olympus, Tokyo, Japan).