Ab973

The prepared femur sections of newborn C57BL/6 mice were subjected to antigen retrieval with heat-induced antigen retrieval solution (ab973, abcam). The specific IF stain procedure was performed as outlined previously 33 (link). Primary antibodies, goat anti-GPR125 (1:200) (Santa Cruz) and mouse-anti-Ctsk (Santa Cruz, sc-48353) were applied to one section. After overnight incubation, secondary antibodies, TRITC donkey anti-Goat antibody (Thermo Scientific) and FITC donkey-anti-mouse antibody (Thermo Scientific) were used to show the specific expression location. Nuclei were visualized with DAPI (1ug/mL).

Slides were baked at 60°C for 1 hr and rehydrated in a gradient ethanol series. Antigen retrieval was performed using the Heat Mediated Antigen Retrieval Solution pH 6.0 (ab973; abcam) following the manufacturer's instructions. Sections were blocked and incubated in primary antibody. The primary antibodies were both polyclonal, raised in rabbits, and used at 1:1000 dilution. The anti‐AMBN antibody was raised against peptide CMRPREHETQQYEYS, which is a remake of the AMBN‐63 antibody (Iwata et al., 2007); the anti‐AMEL antibody was raised against recombinant mouse amelogenin rM179 (Simmer et al., 1994). Sections were incubated in Goat anti‐rabbit IgG(H+L) Secondary Antibody, Alexa Fluor Plus 555 (Invitrogen, A32732) at a concentration of 1:1000, then mounted in Invitrogen^™ ProLong^™ Gold Antifade Mountant with DAPI (P36935; Invitrogen). Pictures were taken using a Nikon SP8 confocal microscope system (Biomedical Imaging Core, University of Michigan).

Frozen sections were fixed in 4% PFA for half an hour and heat-induced antigen retrieval was performed (abcam, ab973). Sections were incubated with mouse IgG blocking solution and universal blocking solution, and then overnight with primary antibodies: anti-YFP rabbit IgG (Santa Cruz sc-410, 1:100) or anti-Gα13 rabbit IgG (Santa Cruz sc-410, 1:100) and anti-Ctsk mouse IgG (Santa Cruz sc-48353, 1:200). Ctsk was then labelled by biotin-anti-mouse IgG (Vector laboratories, BMK-2202) and streptavidin-FITC (sigma). YFP and Gα13 were labelled by TR-goat-anti-rabbit IgG.