Hoechst 33258

The fluorometric assay was performed using a VarioSkan microplate reader (Thermo Fisher, Waltham, MA, USA) in a black 96-well plate. For the DiI (Thermo Fisher, Waltham, MA, USA) and Calcein AM (Abcam, Waltham, MA, USA) assays, cells were pre-stained prior to obtaining vesicles. Cells were washed twice with DPBS, stained with the dyes for 20 min in a CO₂ incubator, washed twice to remove the dyes, and then used for AV induction. A suspension of vesicles from stained cells in DPBS was used for the fluorometric assay. For Hoechst 33258 (Abcam, USA), AVs were first obtained and then stained with Hoechst 33258 for 20 min.

Sections of embryonic brains were thawed for 2 h at room temperature (RT), rinsed in PBS at RT, postfixed in 4% PFA for 5 min, and again rinsed in PBS followed by PBS + 0.5% Triton X-100 (PBT). Then they were incubated in PBT plus 10% normal donkey serum (NDS; Jackson ImmunoResearch) at RT for 2 h, followed by primary antibodies (Table 1) in PBT + 3% NDS at 4°C overnight. After washing in PBT, sections were incubated with secondary antibodies (Table 1) in PBT + 3% NDS for 2 h at RT. Hoechst 33258 (Abcam) was used to counterstain nuclei. Sections of adult brains were rinsed in PBS at RT and then incubated in PBS + 0.5% Triton X-100 (PBT) plus 10% NDS (Jackson ImmunoResearch) at RT for 2 h. Sections were incubated with primary antibodies in PBT + 3% NDS at 4°C overnight. After washing in PBT, sections were incubated with secondary antibodies in PBT + 3% NDS for 2 h at RT. Hoechst 33258 (Abcam) was used to counterstain nuclei.

Gelatin degradation assay was performed as previously described [13 (link)]. In brief, cells in suspension were added to wells containing equilibrated Oregon Green™ 488 conjugate (ThermoFisher Scientific #G13186) gelatin-coated coverslips, either serum-starved combined or not with mβCD for 2 h and then incubated in serum-containing medium for 6–12 h  or serum-starved for 2 h and then incubated in GM6001-supplemented medium for 6–12 h. Cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 15 min, blocked with 5% Normal Goat Serum in PBS for 1 h, all steps at room temperature, immunolabeled with anti-Cortactin (Cell Signaling Technology #3503, 1:200) overnight at 4 °C, and stained with fluorescent secondary antibodies (ThermoFisher Scientific, 1:1000), Alexa Fluor™ 647 Phalloidin (ThermoFisher Scientific #A22287, 1:200) and Hoechst 33258 (Abcam #Ab228550, 1:1000) for 1 h at room temperature in the dark. Coverslips were then mounted with Dako and examined with a Zeiss Cell Observer Spinning Disk (COSD) confocal microscope using a plan-Apochromat 63 × NA 1.4 water immersion objective and the same settings for illumination. Quantification of total gelatin degradation area per total cell area was done using ImageJ/Fiji [13 (link)].