Lsn700

High magnification images were acquired by confocal microscopy (DM IRBE, Leica Microsystems, Zeiss LSN700) from stratum lacunosum-moleculare of CA1 to quantify LEC axonal terminals labeled by BDA. Microscopic stacks were acquired as 2048 × 2048 pixel images (pixel size, 78 nm; Z-step, 500 nm). All images were similarly processed and analyzed using ImageJ software.

Immunofluorescence analysis was performed on 1% Matrigel-coated glass coverslip in wells or in situ in microfluidic channels with the same protocol. Cells were fixed in 4% formaldehyde (Sigma-Aldrich 78775) in PBS for 10 min at RT, washed in PBS, permeabilized for 1 h in PBS + 0.3% Triton X-100 (PBST) at RT, and blocked in PBST + 5% of HS (ThermoFisher 16050-122) for 5 h at RT. Cells were incubated overnight at 4 °C with primary antibodies (see Supplementary Table 2) in PBST + 3% of HS. After washing with PBS, cells were incubated with secondary antibodies (Alexa, Life Technologies) (Supplementary Table 2) for 45 min at RT. Nuclei were stained with either DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich F6057) or Hoechst 33342 (ThermoFisher 62249). In the case of Phalloidin staining (see Fig. 8f), Alexa Fluor 488 Phalloidin and Hoechst were added with secondary antibodies. Images were acquired with a Zeiss LSN700 or a Leica SP5 confocal microscope using ZEN 2012 or Leica TCS SP5 LAS AF (v2.7.3.9723) software, respectively.
For alkaline phosphatase staining, cells were fixed with a citrate–acetone–formaldehyde solution and stained using an alkaline phosphatase detection kit (Sigma-Aldrich 86R-1KT). Plates were scanned using an Epson scanner and scored manually.