Occludin

Washed cultured cells and retinal tissue samples were lysed in modified RIPA buffer supplemented with 1:100 (v/v) of proteinase/phosphatase inhibitor cocktail (Thermo Scientific, Greenville, SC, USA). This was followed by centrifugation at 12,000 xg at 4°C for 30 min to remove insoluble material. Protein was determined by BCA Protein Assay (Thermo Scientific, Greenville, SC, USA) and equal amount of protein was exposed to boiling in Laemmli sample buffer, separated by SDS-PAGE on a gradient gel (4 to 20%, Pierce, Rockford, IL), then transferred to nitrocellulose membrane, followed by incubation with specific antibodies. Antibodies for NMDAR1 (Cell signaling, Danvers, MA, USA, Cat# 5704S), NMDAR2A (Cell signaling, Danvers, MA, USA, Cat# 4205s), NMDAR2B (Cell signaling, Danvers, MA, USA, Cat# 4207s), ZO-1 (Abcam, Cambridge, MA, USA, ab59720), occludin (Invitrogen, Waltham, MA, USA, 71-1500), Albumin (Bethyl, TX, USA), GAPDH, (Sigma-Aldrich, St. Louis, MO, USA) and β actin (Cell signaling, Danvers, MA, USA, Cat# 937215) were detected with a horseradish peroxidase-conjugated antibody and enhanced chemiluminescence (Thermo Scientific, Greenville, SC, USA). Intensity of immunoreactivity was measured by densitometry using Image J software (NIH).

After fixation for 24 hours in 4% paraformaldehyde, plexuses from fetuses at GD120, GD165 and adults (no GD90 animals were available) were dehydrated in increasing concentrations of alcohol and embedded in paraffin. Serial sections were cut on a microtome and prepared for IHC. Paraffin was removed from sections in xylene, sections were rehydrated in decreasing concentrations of ethanol and gently boiled in 0.01 M citrate buffer or protease digestion (Streptomyces griseus, Sigma; 1 mg/ml at 37°C) for 10 min. Blocking steps included H₂O₂ treatment (3% in methanol for 10min) and appropriate serum (5%) or DAKO serum-free protein block for 1 hour. Sections were then incubated in the following primary antibodies towards three key plexus tight-junctional proteins: ZO-1 (1:100; Cat#61-7300, Invitrogen), occludin (1:100; Cat#71-1500, Invitrogen) and claudin-1 (1:200; Cat#71-1500, Invitrogen). They were then incubated in biotinylated secondary against the appropriate species followed by the ABC kit (Vector) or HRP-labelled secondary antibody (BrightVision poly HRP-anti-rabbit IgG from ImmunoLogic) and developed with DAB. In between all steps, sections were washed in PBS with 0.1% tween20. After 5 minute in DAB solution, sections were washed in water, dehydrated and cover-slipped. Blank sections were obtained when primary antibody was omitted.

The cells grown on permeable supports were fixed with absolute methanol and stored at −80°C until used. The cells were thawed, rinsed in PBS, blocked with normal serum, and incubated overnight at 4°C in primary antibody solutions. The cells were washed thoroughly and incubated in secondary antibodies conjugated with fluorescent dyes AF488 or Cy3. Following washings in PBS, the cells were mounted in ProLong Gold antifade reagent (Invitrogen) containing DAPI as a nuclear stain and examined with a Zeiss LSM 510 microscope equipped with a Hamamatsu digital camera (Hamamatsu Photonics, Hamamatsu, Japan). Images were processed with LSM software (Zeis). Primary antibodies used were occludin (Invitrogen, 33-1500), ZO-1 (Invitrogen, 617300), claudin-1 (Invitrogen, 51-9000), claudin-2 (Invitrogen, 51-6100), claudin-4 (Invitrogen, 32-9400), caveolin-1 (Cell Signaling, 3238), CD63 (Santa Cruz Biotechnology, sc-15363) and cavin-1 (Novus, NBP1-80220). Polyclonal Rab5B antibody was a kind gift from Dr. David B. Wilson (School of Medicine, Washington University, St. Louis, MO).