Goat serum

The Plexus choroidei of the lateral ventricles were removed from the brains together with attached ependymal and subependymal tissue and fixed in 4% paraformaldehyde for 24 h. After being rinsed in PBS, the samples were further dissected and placed in 30% (w/v) sucrose for another 24 h for cryoprotection. The fixed samples were frozen in isopentane-nitrogen-cooled TissueTek^® (Sakura, Staufen, Germany) and stored at −80 °C before being cryosectioned at 18 μm.
Next, cryosections were rehydrated and washed in PBS for 10 min before being incubated in blocking solution containing PBS, 4% (v/v) goat serum (Biochrom, Berlin, Germany), 0.1% (v/v) bovine serum albumin (Roth, Karlsruhe, Germany) and 0.1% (v/v) Triton^® X-100 (Roth, Karlsruhe, Germany) for 90 min at room temperature.
Afterwards, sections were incubated with primary antibodies (Table 1) diluted in the preincubation solution overnight at 4 °C in a humidified chamber. After washing with PBS three times for 10 min, the secondary antibodies (Table 1) were applied for 90 min at room temperature. Afterwards, sections were stained with the nuclear stains DRAQ5 (1:1000; Thermo Fisher, Waltham, MA, USA) or DAPI (1:1000) and washed with PBS three times for 10 min before being mounted with Mowiol 4–88 (Roth).

Immunofluorescence studies were performed on culture-plate inserts. Confluent monolayers were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min and permeabilized for 10 min with PBS containing 0.5% (v/v) Triton X-100. To block non-specific binding sites, cells were then incubated in PBS containing 1% (w/v) BSA and 5% (v/v) goat serum (blocking solution; Biochrom) for 60 min. All subsequent washing procedures were performed with this blocking solution. After blocking, cells were incubated 60 min at room temperature with primary antibodies for tricellulin (Abfinity, Invitrogen; 1:250) and occludin (Invitrogen, 1:250), followed by washing steps and incubation during 60 min at room temperature with the respective secondary antibodies (Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 594 goat anti-mouse, each 1:500; Molecular Probes MoBiTec) and 4′,6-diamidino-2-phenylindole (1:1000). Images were obtained with a confocal laser-scanning microscope (LSM 780, Zeiss, Jena, Germany) and processed using ZEN software (Zeiss, Oberkochen, Germany).

To prevent unspecific binding of antibodies, samples were blocked for 30 min with PBS containing 4% (v/v) goat serum (Biochrom, Berlin, Germany), 0.1% (v/v) bovine serum albumin (Roth, Karlsruhe, Germany), and 0.1% (v/v) Triton^® X-100 (Roth, Karlsruhe, Germany), followed by incubation of primary antibodies (Supplementary Table S3) diluted in PBS with 0.1% (v/v) bovine serum albumin and 0.1% (v/v) Triton^® X-100 overnight at 4 °C in a humidity chamber. Afterward, samples were washed with PBS three times for 5 to 10 min. The secondary antibody (Supplementary Table S4) was diluted in PBS, 0.1% (v/v) Triton X-100, and 0.1% (w/v) BSA and incubated for 60 to 90 min at room temperature. Nuclear staining was carried out with 4′,6-diamidino-2-phenylindole (DAPI) or DRAQ5 solution (200 ng/ml; Roth). After two washing steps with PBS for 5 to 10 min, the samples were washed in distilled water for 5 min, followed by mounting with Kaiser’s glycerol gelatine (Merck, Darmstadt, Germany) or Mowiol 4-88 (Roth).