Anti pdgfrβ

Fresh LNs were embedded in tissue-freezing medium. Cryostat sections (8 μm thick) were cut for imaging by fluorescence confocal microscopy. The following primary Abs were used for tissue staining: anti-HEV MECA79 (sc-19602, SCBT), anti-Lyve-1 (ab14917, Abcam), anti-PDPN (AF3244, R&D Systems), anti-ER-TR7 (sc-73355, SCBT), anti-CD11b (101202, BioLegend), anti-PDGFRβ (136005, Biolegend), anti-NG2 (ab129051, Abcam), anti-RANKL (510002 Biolegend), anti-CD35 (NBP2-52667, Novus Biologicals), anti-MAdCAM (16-5997-85,eBioscience), anti-ZO-1 (61-7300, Invitrogen), anti-Collagen I (ab34710, Abcam), anti-Fibronectin (ab45688, Abcam), anti-LepR (L9536, Sigma-Aldrich). The following secondary Abs were used: Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 594-conjugated anti-rabbit IgG, Alexa Fluor 488-conjugated anti-rat IgG, Alexa Fluor 594-conjugated anti-rat IgG, Alexa Fluor 488-conjugated anti-goat IgG, and Alexa Fluor 594-conjugated anti-goat IgG (Jackson ImmunoResearch). The stained tissue sections were imaged using EVOS FL Auto 2 Imaging System (Thermo Fisher Scientific). For the quantification of images, all images were automatically processed using ImageJ (NIH) and split into RGB channels. Auto threshold was used to convert intensity values of the immunofluorescent stain into numeric data. DAPI (VECTASHIELD, Vector Laboratories) was used to stain the cell nuclei.

Flow cytometric analysis of the OP9 cells and primary BM cells were performed with the use of FACSCanto (Becton Dickinson, San Jose, CA). The following antibodies were used: anti-PDGFRβ, anti-CD31, anti-CD45, anti-Ter119, anti-CD54 (Biolegend), anti-CD3, anti-CD4, anti-CD8, anti-CD34, anti-Mac-1, anti-Gr-1, anti-B220, and anti-TER-119, anti-NK.1.1, anti-CD106 anti-hCDRA (BD Biosciences), and anti-CD61 anti-CD140a (eBioscience) anti-CD44 anti-hCD45RO (Pharmingen), anti-Notch-1, anti-Notch-4, anti-Jagged-1 (eBioscience), anti-Notch-2, anti-Notch-3, anti-Dll-1, and anti-jagged-2 (Biolegend).