Carvii confocal imager

Cells, 1.0 × 10⁵, were seeded and grown as a monolayer on 8‐well chambered slides (LAB‐TEK) for 3 weeks. Cells were washed with PBS and fixed with one of the following steps: (1) preextracted with 0.2% TritonX/PBS on ice followed by 1% paraformaldehyde/PBS for 15 min at RT; (2) fixed directly with 1% paraformaldehyde/PBS for 15 min at RT; (3) fixed directly with 100% methanol for 5 min on ice; and (4) fixed directly with 100% Acetone for 5 min on ice. Cells were permeabilized with 0.5% Tween/PBS and blocked in 0.1% Tween/casein/PBS before primary antibody staining. Tissue postfixation was done with 4% paraformaldehyde/PBS and mounted with Prolong Gold antifade reagent with DAPI (Life Technologies). Images were obtained using a spinning disk BD CARVII Confocal Imager (BD Biosystems, San Jose, CA) on a Zeiss Axio Observer inverted microscope (Carl Zeiss Microscopy, Thornwood, NY) at 63X objective lens controlled by MetaMorph imaging software (Molecular Devices, Sunnyvale, CA). Image Z resolution was further optimized with Volocity software (PerkinElmer, Waltham, MA). For non‐Z‐stacked higher resolution images, a Leica SP5 inverted laser confocal micorscope was used. Antibodies used: Anti‐claudin 3(#ab15102, AbCam, Cambridge, MA), anti‐claudin4 (#329400, Life Technologies), Anti‐human CD137 (#552533, BDBioscience), phalloidin‐Alexa 647(A22287, Life Technologies).