Pe conjugated rat anti mouse cd45 30 f11

PαS cells, which co-expressed PDGFRα and Sca-1, but not CD45 and TER119, were isolated from the bone of WT or OSMR KO mice as described by Morikawa et al. [10] (link). Briefly, bone marrow was flushed out from femurs and tibias of 8–12 mice using 22- and 23-G needles (Terumo) and the remaining bone was ground with a pestle. Then, the crushed bone fragments were treated with 0.2% collagenase (Sigma) for 30 min at 37°C. Next, after depletion of mature red blood cells using hypotonic lysis buffer (0.38% NH₄Cl) for 7 min on ice, the cells were incubated with anti-FcR for 15 min on ice. The cells were then incubated with allophycocyanin (APC)-conjugated rat anti-mouse PDGFRα (APA5; eBioscience, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse Sca-1 (E13-161.7; BD Biosciences, Franklin Lakes, NJ, USA), phycoerythrin (PE)-conjugated rat anti-mouse CD45 (30-F11; BD Biosciences), and PE-conjugated rat anti-mouse TER119 (TER-119; eBioscience). After dead cells were excluded by propidium iodide staining, the CD45^- TER119^- PDGFRα⁺ Sca-1⁺ cells were sorted using a Moflo XDP cell sorter (Beckman-Coulter, Fullerton, CA, USA). MSC were sub-cultured in α-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (JRH Biosciences, Inc., Lenexa, KS, USA), penicillin-streptomycin-glutamine solution (Invitrogen), and Gluta-Max (Invitrogen).

From PBS-perfused mice, tissue was fixed with 4% formaldehyde by perfusion or ex vivo and cryopreserved in 30% sucrose in PBS. Ten-micrometer sections were prepared using a Leica CM1860 UV cryostat at −22 to 25°C. Tissue was mounted onto Colorfrost Plus microscope slides (Fisher Scientific). Sections were blocked with 5% normal goat serum (NGS) or 0.1% fish skin gelatin (FSG) and 0.1% Triton X-100 in PBS. Primary antibodies were incubated overnight at 4°C, and secondary antibodies were incubated for 1 to 2 h at room temperature in the dark. The antibodies and dilutions used were Alexa Fluor 488-conjugated F(ab′)₂ fragment of goat anti-mouse (H+L) at 1:250 (Life Technologies, Inc.), Alexa Fluor 488-conjugated goat anti-mouse (H+L) at 1:250 (Life Technologies, Inc.), Alexa Fluor 488-conjugated AffiniPure goat anti-human IgG (H+L) at 1:250 (Jackson), phycoerythrin (PE)-conjugated rat anti-mouse CD45 (30-F11) at 1:100 (BD Pharmingen), PE-conjugated anti-mouse CD4 (L3T4) at 1:200 (EBioscience), Alexa Fluor 594-conjugated rat anti-mouse CD31 (MEC13.3) at 1:200 (BioLegend), and Alexa Fluor 555-conjugated goat anti-rabbit (H+L) at 1:250 (Life Technologies, Inc.). Microscopy images were acquired on an Axio Observer Z1 microscope with a motorized stage (Zeiss) with an EC Plan-Neofluar 10×/0.3 objective (Zeiss), and tiled images were stitched and processed using Zen Blue (Zeiss).