Mouse anti human cd34

Synovial membranes from intact areas of clinically normal joints were harvested and subjected to sequential digestion using 1.2 U/ml dispase and 112 U/ml type I collagenase to isolate MSCs, as previously described[1 (link), 8 (link)]. Monolayer cultures of MSCs from synovial membrane were cultured in Dulbecco´s Modified Eagles Medium (DMEM), 15% v/v foetal bovine serum (FBS), 1% v/v penicillin and 1% v/v streptomycin (all from SIGMA-ALDRICH, Missouri, USA), when the cells lead 90% of confluence in culture they were separated into CD-105⁺sub-population by FACSAria cytometry sorter (BD Bioscience, Madrid, SP) using an antibody against anti-human CD-105 (BD, Pharmigen), which dilution was 1:20 of antibody each 1x10⁶ cells in 200 μl of PBS. The CD-105⁺sub-population were characterized by flow cytometry. The primary antibodies used were mouse anti-human CD34 (1:20 DakoCytomation, Barcelone, SP), FITC mouse anti-human CD45 (1:20), FITC mouse anti-human CD105 (1:100 from Serotec, Bavaria, GER), FITC mouse anti-human CD44 (1:100 from Serotec, Bavaria, GER) and PE-Cy5-conjugated mouse anti-human CD90 (1:20 from BD Pharmagen, Madrid, SP). Flow cytometry data were generated on CellQuest and DIVA software (BD Bioscience, Madrid, SP).

For immunofluorescence studies, specimens were fixed for 20–25 min in 4% paraformaldehyde (PFA), rinsed in PBS, transferred into 10% and 30% sucrose in PBS, and embedded in tissue freeze medium (Tissue Tek, Sakura Finetek Zoeterwoude, NL). Sections of 12–14μm were incubated with the following primary antibodies: Rabbit-anti-human CCBE1 (1:500, Sigma-Aldrich, München, Germany), rabbit-anti-human ESAM-1 (1:100, Sigma-Aldrich, München, Germany), goat-anti-human smooth muscle α-actin (SMA) (Acris Antibodies, Herford, Germany), mouse-anti-human CD31 (1:50, BD Pharmingen), mouse-anti-human CD117 (c-KIT, 1:100, Santa Cruz Biotechnol., and 1:1, Dako, Hamburg, Germany), mouse-anti-human D2-40 (1:200, Dako, Hamburg, Germany), rabbit-anti-human Lyve-1 (1:500, ReliaTech, Braunschweig, Germany), rabbit-anti-human Prox1 (1:500, ReliaTech, Braunschweig, Germany), mouse-anti-human vimentin (1:200, Dako), mouse-anti-human PDGFRα (1:1, Dako), mouse-anti-human CD34 (1:200, Dako). Secondary antibodies were: goat-anti-mouse Alexa 488/594, goat-anti-rabbit Alexa 594, donkey-anti-goat Alexa 488 (MobiTech, Göttingen, Germany). Sections were counter-stained with Dapi and mounted under cover slips with Fluoromount-G (Southern Biotechnology, US). Photos were taken with AxioImagerZ1 (Zeiss, Göttingen, Germany).