Cd140a

The tissue sections were dried at RT for 20 minute and then blocked with 1% BSA PBS for 3×10 minutes, the sections were incubated with primary antibody at 4 °C overnight. The sections were washed with 1% BSA PBS buffer and then incubated with the secondary antibody conjugated with Alexa-dye for 1 hour at RT in dark room. The sections were washed with 1% BSA PBS buffer and stained with 0.1% DAPI for 5 minutes at RT in dark room. The sections were then washed and mounted in anti-fade mounting media (Vector Laboratories). The stained slides were observed using an Olympus IX81 microscope or scanned using an iCys Research imaging cytometer (CompuCyte).
The following antibodies were used in this study: Col2 (1:100, Cat. ab34712, Abcam), osteocalcin (1:200, Cat. Ab14173, Abcam), Kitl (1:100, Cat. sc-13126, Santa Cruz), CXCL12 (1:100, Cat. sc-6193, Santa Cruz), α-SMA (1:100, Cat. ab5694, Abcam), CD146 (1:100, Cat. 134702, Biolegend), CD140a (1:100, Cat. 135902, Biolegend), Sca-1 (1:100, Cat. 108102, Biolegend), CD31 (1:150, Cat. 5550274, BD). The secondary antibodies conjugated with Alexa fluor 488 and Alexa fluor 555 were from Invitrogen. In study with mouse primary antibody, the M.O.M. immunodetection kit (Cat. BMK-2202, Vector Laboratories) was used to eliminate the endogenous mouse immunoglobulins background in the tissue.

Cells were stained with antibodies diluted in fluorescence-activated cell sorting (FACS) buffer (DMEM/0.1% sodium azide/1% FCS). Antibody specific for FcγII/IIIR (CD32/CD16) (eBiosciences: San Diego, CA, USA) was used at 5 μg/10⁶ cells in 1 ml to block nonspecific antibody binding to cells. Fluorochrome or biotin-conjugated antibodies for CD11b, CD11c, MHC-II, F4/80, CD3, B220, CD150, CD48, Ly6G, CD45.2, CD29, CD51, CD54, CD31, gp38, CD105, Thy1.2, Sca-1, VCAM1, CD140a, Flt3, NK1.1, CD19, Gr-1, Ter119, and c-Kit, as well as streptavidin-PE-Cy7, streptavidin-PE, and streptavidin-FITC, were purchased from BioLegend (San Diego, CA, USA). Staining with 1 μg/ml propidium iodide (PI) (Sigma-Aldrich) was used to discriminate live cells. Isotype-matched control antibodies were used to set gates to assess specific antibody binding. Median fluorescence intensity (MFI) was calculated as the net change in median channel fluorescence for specific antibody above isotype control. Fluorescence minus one (FMO) controls were used to set gates for specific antibody binding in multicolour staining experiments. Flow cytometric analysis was carried out using BD FACSDiva (Becton Dickinson) and FlowJo software (Tree Star: Ashland, OR, USA).

Pancreatic islets were isolated as previously described [21 ]. Mice were euthanised by cervical dislocation. The pancreas was inflated with 3 ml cold collagenase (Sigma; St Louis, MO, USA) solution (0.3 mg/ml) through the bile duct with a 20G needle starting at the gall bladder. The pancreas was then removed into a siliconised glass tube containing 2 ml of 1 mg/ml collagenase solution and digested at 37°C in a water bath for 12–15 min. After three washes of the digested pancreas, islets were hand-picked and counted under a dissecting microscope for further experiments. For single-cell isolation, the islets were treated with Cell Dissociation Solution (Sigma) and the single-cell suspension was harvested. Beta cells from the dissociated islets were stained with fluorochrome-conjugated monoclonal antibodies to CD45 (BioLegend; San Diego, CA, USA), CD140a (BioLegend) and FluoZin-3-acetoxymethyl (AM) (CD45⁻FluoZin-3-AM⁺; ThermoFisher, Waltham, ME, USA) [22 ] before being analysed by flow cytometry (LSRII; BD Bioscience, San Diego, CA, USA).

The following antibodies were used in flow cytometry: phycoerythrin (PE)-conjugated anti-mouse CD44 (Biolegend, 103007), Sca-I (eBioscience, 12-5981-83), CD140a (Biolegend, 135906), CD13 (BD Pharmingen, 558745), MHC II (eBioscience, 12-5320-80), CD11b (eBioscience,12-0112-83), CD11c (eBioscience, 12-0114-82), CD86 (eBioscience, 12-0862-82), and CD45 (eBioscience, 12-0451-82). Antibodies used for western blotting analysis were: anti-p53 (FL-393; sc-6243, Santa Cruz Biotechnology); GAPDH (14C10, 2118, Cell Signaling Technology). Antibody used for Chip was: p53 (FL-393; sc-6243, Santa Cruz Biotechnology), IgG (normal mouse IgG, sc2025, Santa Cruz Biotechnology). The reagents for cell treatment were: Nutlin-3 (Nutlin-3, Selleck Chemicals, Houston, TX, USA), Cisplatin (Sigma-Aldrich), RANKL (recombinant mouse RANKL, 50 ng/ml, R&D), M-CSF (recombinant mouse M-CSF, 25 ng/ml, R&D), and OPG (recombinant OPG, 50 ng/ml, Sigma-Aldrich). RANKL for mouse injection (recombinant mouse RANKL, 2 mg/kg/day) was from R&D Systems.