Af2910

Bone marrow cells were harvested from bilateral femur of the mice. Goat polyclonal alkaline phosphatase (ALP) primary antibody (1:50, R&D systems, AF2910) and phycoerythrin (PE)-conjugated anti-goat IgG secondary antibody (1:200, R&D systems, F0107) were used to stain the ALP⁺ populations. Briefly, after washing with PBS and incubating with 1% BSA, the bone marrow cells were stained with the Anti-Mouse ALP polyclonal antibody. Then, the stained cell populations were washed three times and subsequently incubated with PE-conjugated secondary antibody. Finally, the incubated cells were washed, then sorted and analysed by FACS Aria II Flow Cytometer (BD Biosciences). The selected ALP⁺ cells were used for total RNA extraction followed by real-time PCR analysis44 (link)45 (link).

All decalcified specimens in the 4-week and 12-week groups detected the expression of alkaline phosphatase (ALP) by immunofluorescence staining. The primary antibody (anti-ALP 1 : 50, AF2910) was purchased from R&D Systems (USA). The secondary antibody conjugated to Alexa Fluor® 488 (1 : 200, Thermo Fisher, USA) was used. After washing secondary antibody three times by PBS, the cell nuclei were stained by DAPI (1 : 200, Thermo Fisher, USA). When we finished the examination of ALP expression in all specimens, the slices were stained with hematoxylin-eosin (HE) for general histology. The amount of the positive expression was analyzed using a fluorescent microscope (Olympus Corporation, Japan) with higher magnifications (200×) and counted on five random fields for each section by a blinded investigator to experimental design. Data was analyzed by ImageJ software.

The deparaffinized sections were subjected to antigen retrieval, incubated in 3% hydrogen peroxide for 10 min, blocked in PBS with 10% normal goat serum and 10% Triton X-100 for 1 hour, and then stained overnight at 4 ℃ with mouse anti-LepR (catalog number MAB867, 1:100, R&D Systems) or rabbit anti-CD55 (ab133684, 1:100, Abcam) or mouse anti-collagen I (ab88147, 1:100, Abcam) or rabbit anti-CD358 (ab198034, 1:20, Abcam) or rabbit anti-CXCL12 (97958, 1:200, Cell Signaling Technology) or goat anti-ASPN (ab31303, 1:25, Abcam) or rat anti-CD31 (NB600-1475SS, 1:100, Novus biologicals) or goat anti-ALP (AF2910, 1:100, R & D Systems) or mouse anti-EphrinA1 (sc-377362, 1:200, Santa Cruz) or rabbit anti-Eph A7 (PA1-30296, 1:200, Invitrogen). After rinsing with PBS for 15 minutes, the tissues were incubated at room temperature with goat anti-rabbit Alexa Fluor 488 or goat anti-mouse Alexa Fluor 568 or goat anti-rabbit Alexa Fluor 568 or goat anti-mouse Alexa Fluor 488 or donkey anti-rat Alexa Fluor 405 or goat anti-rabbit Alexa Fluor 647. Slides were mounted with mounting medium containing DAPI (Vector Labs), and images were captured using a Leica DM4000 fluorescence microscope.