Dylight 649 donkey anti rabbit igg

Laser‐scanning cytometry was performed as described earlier (Nombela‐Arrieta et al., 2013). Briefly, the mice were perfused post‐mortem with 10 ml paraformaldehyde‐lysine‐periodate (PLP) fixative through the vena cava to achieve rapid in situ fixation and optimal preservation of the BM tissue. Femoral bones were isolated, fixed in PLP for 4–8 hr, rehydrated in 30% sucrose/phosphate‐buffered saline (PBS) for 48 hr and snap frozen in OCT (TissueTek). Cryosections of non‐decalcified whole longitudinal femoral bones were obtained using a Leica Cryostat and the Cryojane tape transfer system (Leica Microsystems). BM sections were stained with rabbit anti‐Laminin (Sigma Aldrich; L9393) and goat anti‐c‐kit (R&D systems; AF1356) polyclonal antibodies. As secondary antibodies, DyLight 488‐donkey anti‐goat immunoglobulin G (IgG) and DyLight‐649 donkey anti‐rabbit IgG (Jackson Immunoresearch) were employed. 4′,6‐Diamidino‐2‐phenylindole (DAPI; Invitrogen) staining was used for nuclear detection and sections were mounted with Vectashield mounting medium for immunofluorescence (Vector Labs). 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 PMT detectors with bandpass emission filters at 450/40, 521/15, 575/50, and 650LP.