Eclipse t

Transwells were cut from the chamber, cut in half with a sharp blade, and cells were permeabilized with 0.1% Triton X-100 for 15 min, then blocked with 0.1% BSA in PBS for 1 h.⁴⁴ (link) Primary antibodies were diluted in 0.1% BSA in PBS and incubated with cells overnight at 4°. Primary antibodies used were as follows: ZO-1 (1:100, 61-7300, Invitrogen, Carlsbad, CA, USA), V5 (1:50, R96025, Invitrogen, Carlsbad, CA, USA), ARL13B (1:100, 17711-1-AP, Proteintech, Rosemont, IL, USA), COL IV (1:100, AB6586, Abcam, Cambridge, MA, USA), and COL VI (1:100, AB6588, Abcam, Cambridge, MA, USA). Secondary antibody was diluted 1:500 in 0.1% BSA in PBS and incubated with cells for 2 h at room temperature. Secondary antibodies used were labeled with Alexa Fluor 488 (A11034, Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 555 (A21429, Thermo Fisher Scientific, Waltham, MA, USA), or Alexa Fluor 647 (A21235, Thermo Fisher Scientific, Waltham, MA, USA). Lastly, cells were incubated with DAPI, rinsed with PBS, and mounted with Fluoromount-G (17984-25, Electron Microscopy Sciences, Hatfield, PA, USA). Samples were imaged with a TCS SP5 II confocal laser scanning microscope (Leica, Allendale, NJ, USA) or fluorescence microscope (Eclipse T, Nikon, Melville, NY, USA).