Ab7754

Immunohistochemistry was used to detect the expression of specific intestinal markers within the SMI microtissues including cytokeratin (CK)-19 (marker for epithelial cells), ZO-1 (marker for tight junctions), villin (marker for brush borders), and vimentin (marker for fibroblasts). 5-μm-thick paraffin sections were mounted on glass slides, deparaffinized, and rehydrated using a graded series of ethanol. Slides were processed for antigen retrieval by heating in a citrate buffer (pH 7.4) for 45 min at 95–99°C. After 45 min, the specimens were removed from the water bath and left in the citrate buffer for an additional 20 min at room temperature (RT). Antibodies for the cytokeratin CK19 (2 μg/mL, cat# ab7754, Abcam, Cambridge, MA), tight junction protein, ZO-1 (1 μg/mL, cat# 61–7300, Life Technologies, Carlsbad, MA), brush border marker, villin (2 μg/mL, cat# ab97512, Abcam, Cambridge, MA), and fibroblast marker, vimentin (50 μg/mL, cat# ab24525, Abcam, Cambridge, MA), and p-glycoprotein (P-gp; Abcam Catalogue# ab170904) were used. Primary antibodies were applied for 60 min at RT. Antibodies were visualized using AlexaFluor® secondary antibodies (5 μg/mL, Life Technologies, Carlsbad, CA). DAPI (cat# D1306, Life Technologies, Carlsbad, CA) was used for nuclear staining. All images were captured using a FV1000 confocal microscope (Olympus America Inc., Center Valley, PA).