Claudin 1

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.

Plasmids were transfected into HEK293a or Schwann cells using Effectene (Qiagen). Supernatants from the transfected cell lysates and corresponding antibodies were used for co-IP, and were incubated with protein-G agarose beads (Life Technologies). Proteins were boiled off the beads in 2× SDS loading buffer and separated on SDS–PAGE for Western blot analysis. The following antibodies were used: ZO₁ (Invitrogen #61-7300 for IP; #339100 for Western blot), claudin-1 (Invitrogen, #37-4900).

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).