Trpv6

Ileal tissue or cell samples were added to RIPA lysate buffer containing 1% protease inhibitor (Beyotime) to extract total protein. The primary antibodies against Occludin (Proteintech, dilution ratio 1:1000), ZO-1 (Proteintech, dilution ratio 1:1000), TRPV6 (Proteintech, dilution ratio 1:1000), KLB (Aviva Systems, dilution ratio 1:1000), FXR1 (Proteintech cat#13194-1-AP, RRID: AB_2110702, dilution ratio 1:1000), HNF4A (Abcam cat#ab181604, RRID: AB_2890918, dilution ratio 1:1000), Actin (Proteintech, dilution ratio 1:2000) were used. The appropriate HRP-conjugated secondary antibodies (Proteintech, dilution ratio 1:4000) were utilized after overnight incubation of the primary antibodies at 4°C. The antibody validation was provided in the supplemental materials.

Immunocytochemistry was conducted as previously described (Pediaditakis et al., 2021 (link)). Cells were blocked on the Brain-Chip in phosphate-buffered saline (PBS) containing 10% donkey serum (Sigma) at 4°C overnight. Saponin 1% was used to permeabilize membrane when required. Primary antibodies were MAP2 (Thermo Fisher Scientific; MA512826), VGLUT1 (Thermo Fisher Scientific; 48-2400), Synaptophysin (Abcam; 32127), GFAP (Abcam; ab53554), GLAST (Invitrogen; PA5-19709), s100β (Abcam; 52642), NG2 (Abcam; ab83178), αSMA (Abcam; 7817), IBA1 (FUJIFILM; 019-19741), CD68 (Abcam; ab213363), ICAM-1 (R&D Systems; BBA3), Ki67 (Abcam; 197234), ZO-1 (Thermo Fisher Scientific; 402200), Occludin (Invitrogen; OC-3F10), Claudin-4 (Invitrogen; 329494), TRPV6 (Proteintech;13411-1-AP), PECAM1 (Thermo Fisher Scientific; RB-1033-P1), CD11b (Invitrogen; MA1-80091), CD45 (Invitrogen; 17-0409-42), GLUT1 (Thermo Fisher Scientific; SPM498), P-gp (Thermo Fisher Scientific; MA5-13854), MRP-1 (Millipore; MAB4100), Transferrin receptor (Abcam; 216665). Chips treated with corresponding Alexa Fluor secondary antibodies (Abcam) were incubated in the dark for 2 h at room temperature. Cells were then counterstained with nuclear dye DAPI. Images were acquired with an inverted laser-scanning confocal microscope (Zeiss LSM 880).