Rabbit anti ly6g

LyzM-eGFP mice were perfused postmortem with 10 ml paraformaldehyde-lysine-periodate fixative through the vena cava to achieve rapid in situ fixation and optimal preservation of the BM tissue. Femoral bones were isolated, fixed in paraformaldehyde-lysine-periodate for 4–8 h, rehydrated in 30% sucrose/PBS for 48 h, and snap frozen in optimal cutting temperature compound (TissueTek). Cryosections of nondecalcified whole longitudinal femoral bones were obtained using a cryostat (CM3050S; Leica Biosystems) and the Cryojane tape transfer system (Leica Biosystems). After blocking with normal serum, the slides were incubated with primary antibody (rabbit anti-Ly6G from Abcam) for 30 min at room temperature. Next, the slides were washed three times with PBS followed by incubation with Texas red–conjugated goat anti–rabbit IgG secondary antibody (Thermo Fisher Scientific). DAPI (Invitrogen) staining was used for nuclear detection, and sections were mounted with Vectashield mounting medium for immunofluorescence (Vector Laboratories). High-resolution images of whole longitudinal immunostained femoral sections were obtained with an iCys Research Imaging cytometer (Compucyte Corporation) equipped with four laser lines (405, 488, 561, and 633 nm) and four photomultiplier tube detectors with bandpass emission filters at 450/40, 521/15, 575/50, and 650LP.