Alexa fluor 488 or 568 labeled secondary antibody

Cells were seeded onto fibronectin coated 13-mm glass coverslips (5 μg/mL fibronectin) and then incubated for 24 h. Afterwards cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.5% Triton in PBS with 1% BSA, 100 μg/mL gammaglobulin and 0.05% sodium azide. Cells were blocked for 30 min with 5% BSA in PBS with 100 μg/mL gammaglobulin and 0.05% sodium azide and stained with primary antibodies against β-catenin (BD Transduction Laboratories) or zonulin (ZO)-1 (Thermo Fisher Scientific) followed by Alexa Fluor 488 or 568-labeled secondary antibodies (Life Technologies). Cells were mounted in Prolong Gold (Invitrogen) and imaged with a Leica DMR 020–525.024 fluorescence microscope and Leica immersion objective HCX PL APO 40×/1.25–0.75. Images were collected using Leica TCS software.

Sections (5 µm) of fetal brain and spinal cord were dried, permeabilized with 0.1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) in PBS and blocked for 1 h in 5% BSA at RT. Sections were incubated with primary antibody; anti-GFAP (1:1000, DAKO, Carpinteria, CA, USA), anti-Iba1 (1:1000, Wako, Richmond, VA, USA), anti-p21 (1:500, Abcam, Cambridge, MA, USA), anti-Cleaved Caspase 3 (1:500, Cell Signalling, Danvers, MA, USA), overnight at 4 °C in a humid chamber. Slides were washed and incubated for 1 h with Alexa Fluor 488 or 568 labeled secondary antibodies (1:1000, Life Technologies, Eugene, OR, USA) at RT in the dark. Slides were washed and mounted in mounting media with DAPI (SouthernBiotech, Birminghan, AL, USA).