Anti human fibronectin

hPDLSCs placed on CTRL and TEST samples were fixed for 10 min at room temperature (RT) with 4% paraformaldehyde in 0.1 M PBS, pH 7.4. After PBS wash, cultures were made for immunofluorescence labelling. Then, cells seeded on granules were permeabilized with 0.5% Triton X-100 in PBS, followed by blocking with 5% skimmed milk in PBS. Primary monoclonal antibodies to anti-human Fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Laminin (Santa Cruz Biotechnology), N-Cadherin (Santa Cruz Biotechnology) and RUNX2 (Santa Cruz Biotechnology) were utilized, followed by Alexa Fluor 488 green fluorescence conjugated goat anti-mouse as secondary antibodies (Molecular Probes, Invitrogen, Eugene, OR, USA). Then, the specimens were incubated with Alexa Fluor 594 phalloidin red fluorescence conjugate (Molecular Probes) to stain actin cytoskeleton. Nuclei were dyed with TOPRO (Molecular Probes). Specimens were positioned facing down on glass slides and mounted with Prolong antifade (Molecular Probes) [27 (link)]. The stained samples were evaluated using a Zeiss LSM800 META confocal system, connected to an inverted Zeiss Axiovert 200 microscope equipped with a Plan Neofluar oil-immersion objective (40×/1.3 NA). The images were taken using an argon laser beam with excitation lines at 488 nm.

It should be noted that all experiments were performed with cells cultivated from surgical dissected tumors described above. Primary human meningioma cells were cultured in glutamine-supplemented Dulbecco’s modified Eagle’s medium (DMEM) plus 20 % fetal calf serum (FCS) as described previously [15 (link)]. Subcultures from 2 to 4 were used. Meningiomas express both mesenchymal and epithelial markers of which EMA (epithelial membrane antigen) and fibronectin can be detected in 90 to 100 % of all investigated solid meningiomas, depending on marker and investigated cohort [16 (link)–18 (link)]. Thus, identity and purity of meningioma cell cultures were proven routinely by fibronectin (1:100; rabbit polyclonal anti-human fibronectin; Santa Cruz Biotechnology, Santa Cruz, CA), EMA (1:20; mouse monoclonal anti-human EMA; DAKO, Glostrup, Denmark), and glial fibrillary acidic protein (GFAP) (1:200; mouse monoclonal anti-human GFAP, DAKO) immunocytochemistry staining. The primary antibody was omitted for negative controls. All cultured meningiomas showed a positive staining for EMA and fibronectin, but lack GFAP expression.