Appropriate secondary fluorescently labeled antibodies

Briefly, 4% PFA was used to fix human surgical glioma specimens overnight. Samples were blocked with 10% normal donkey serum supplemented with 0.2% Triton X-100 in PBS for 30 min at room temperature and then incubated with primary antibodies overnight at 4°C, followed by incubation with appropriate secondary fluorescently labeled antibodies (Invitrogen) for 2 h at room temperature. Nuclei were counterstained with Hoechst. Two GBM patients’ clinical specimens were used in this study.

Immunofluorescent staining of cells was performed as previously described^{25 (link)}. Briefly, GSCs were plated on 1% Matrigel covered coverslips overnight before fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 10 min. Samples were permeabilized with 0.2% Triton X-100 (Bio-Rad) in PBS for 20 min and blocked with 10% BSA (Sigma-Aldrich) for 60 min at room temperature. Primary antibody incubation was carried out overnight at 4 °C followed by appropriate secondary fluorescently labeled antibodies (Invitrogen) for one hour at room temperature. Nuclei were counterstained with DAPI. Images were acquired using a wide-field fluorescence microscope or SP-5 confocal microscope (Leica).

Senescent fibroblasts were incubated with an FBS-free medium in the presence of EVs or CS-EVs for 24 h. PBS and CS served as the control. After washing by PBS, the cells were fixed with 4% formaldehyde (Sigma-Aldrich) and then blocked with 10% BSA for 2 h. Then, the cells were incubated with the primary antibody against Ki67 (Abcam, Cambridge, MA, USA) at 4 °C overnight, followed by appropriate fluorescently labeled secondary antibodies (Life Technologies, Carlsbad, CA). Nuclei were stained with DAPI (Sigma) for 5 min. The number of Ki67-positive cells in three random fields in each group was measured by ImageJ software.

8–10 weeks old male C57Bl/6 mice were treated by i.p. injection with 1 mg per mouse of neutralizing anti-CSF1 antibody (Clone 5A1, BioXCell, USA) or isotype control (BE0088, BioXCell, USA) and 4 days later with 0.5 mg/mouse antibody given i.p. as described by DeNardo et al [22] (link). At day 7 after initiation of treatment animals were administered radiolabeled HRG for biodistribution analysis. Blood was taken from control animals prior to treatment, at 3 and 7 days after treatment, for endogenous HRG analysis. After 7 days, organs were harvested for immunofluorescent analysis using anti-CD115 (sc-692, Santa Cruz Biotechnology, USA), anti-CD68 (MCA1957AbD Serotec, USA), anti-Ly6G (551459, BD Biosciences, USA), and appropriate fluorescently labeled secondary antibodies (Life Technologies). Immunoblotting to show plasma protein levels was done using anti-Fibrinogen (GAM/Fbg/7S, Nordic Immunology, The Netherlands) and anti-VWF (A0082, Dako, USA) antibodies.