Rabbit anti scf polyclonal

Bone sections were permeabilized in PBS containing 0.5% Triton X-100 at RT for 20 min and blocked with 5% BSA in PBS at RT for 1 h. The sections were then stained with primary antibodies against DAPI (Invitrogen), anti-VE-cadherin (R&D, 162709), anti-CD31 (BD, MEC 13.3), anti-SDF-1α (R&D, 79018), anti-endomucin (Abcam, V.7C7.1), rabbit anti-SCF polyclonal (Abcam), and goat anti-LepR-biotin polyclonal (R&D Systems) overnight at 4 °C. Sections were then washed with PBS and incubated with Alexa Fluor 488-conjugated donkey anti-rat IgG (Invitrogen), Alexa Fluor 546-conjugated donkey anti-rabbit IgG (Invitrogen), DyLight 488-conjugated streptavidin (BioLegend), or DyLight 647-conjugated streptavidin (BioLegend) at RT for 1 h. Fluorescence images of all samples were captured on an LSM 710 confocal microscope (Carl Zeiss). To measure the number of SCF⁺ cells, we counted LepR⁺SCF⁺ cells attached to endothelial cells.

Enzymatically digested BM cells were seeded in eight-well Lab-Tek II Chamber slides (5 × 10⁴ cells/well, Sigma-Aldrich). Cells were cultured in α-MEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) for 3 days and then treated with sodium l-lactate (5 mM, Sigma-Aldrich), 3,5-DHBA (0.2 mM, Sigma-Aldrich) or 3-OBA (3 mM, Sigma-Aldrich). Plated MSCs were fixed with Cytofix/Cytoperm (BD) for 20 min at 4 °C and subsequently washed with Perm/Wash buffer (BD) during immunocytochemistry. After blocking with 5% BSA in PBS for 1 h, MSCs were stained with DAPI, goat anti-LepR-biotin polyclonal (R&D Systems), anti-endomucin (Abcam, V.7C7.1), anti-SDF-1α (R&D, 79018), and rabbit anti-SCF polyclonal (Abcam) antibodies overnight at 4 °C. After one wash with Perm/Wash buffer, cells were stained with DyLight^TM 488-conjugated streptavidin (BioLegend) or Alexa Fluor 546-conjugated donkey anti-rabbit IgG (Invitrogen) as secondary antibodies. All samples were viewed under a confocal laser scanning microscope (Carl Zeiss).