Anti mouse ly6g antibody

Neutrophil depletion with Ly6G antibody was carried out as previously described (30 (link)). Briefly, WT and IP6K1-deficient mice were intraperitoneally injected with a single dose of anti-mouse Ly6G antibody (400 μg/kg; clone 1A8, BioLegend). The antibody was administered intraperitoneally to obtain a sustained depletion over the first 48 hours of the experiment. Differential white blood cell count using Wright-Giemsa staining was performed to confirm that the neutrophil depletion was successful (peripheral blood neutrophil count was reduced by >85%) (30 (link)).

Paraffin-embedded sections of mouse kidney tissue fixed in 10% formalin were cut at 5-µm thickness, and deparaffinized. Hematoxylin and eosin staining and Sirius red staining (#24901, Polysciences) were performed according to manufacturer’s protocol. The degree of tubulointerstitial damage was scored as previously described^{41 (link)}. The sections were semi-quantitatively scaled (0 to 5+), according to the percentage of the area affected by hyaline casts, tubular atrophy, tubular lumen dilation, and interstitial immune cells infiltration (0 = normal; 1 = <10%; 2 = 10–25%; 3 = 26–50%; 4 = 51–75%; 5 = >75%). 5–8 independent fields were analyzed, and the mean value was plotted.
For immunofluorescence staining, sections were incubated with citrate buffer at 95°C for 10 minutes for antigen retrieval. Sections were allowed to cool slowly for 1 hour, and washed in distilled water, Non-specific signal was blocked with 10% FBS at room temperature for 1 hour. Sections were incubated overnight at 4°C with FITC-labelled anti-mouse Ly-6G Antibody (BioLegend, #127605). Sections were mounted with ProLong^® Gold Antifade Mountant with DAPI (#P36935, Invitrogen) and examined under a fluorescence microscope (OLYMPUS DP73) . The Ly6G+ cells were counted in 5–8 independent fields in kidneys from each mouse (n = 4, each group), and the mean value was plotted.