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Immunohistochemical detection of tight junction proteins

TS Tanvi Shinde
AP Agampodi Promoda Perera
RV Ravichandra Vemuri
SG Shakuntla V. Gondalia
DB David J. Beale
AK Avinash V. Karpe
SS Sonia Shastri
WB Waheedha Basheer
BS Benjamin Southam
RE Rajaraman Eri
RS Roger Stanley
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Immunohistochemical detection of epithelial tight junction (TJ) proteins: ZO-1, occludin and claudin-1 was performed using a Rabbit specific HRP/DAB (ABC) Detection IHC kit (ab64261, Abcam, Australia) following the manufacturer’s instructions and as previously described [19]. Antibodies anti-ZO-1 (NBP1-85046, Novus Biologicals, Australia, 1:400); anti-occludin (NBP1-87402, Novus, 1:600) and anti-claudin-1 (NBP1-77036, Novus, 1 μg/mL) were used for incubating the colonic sections overnight at 4 °C. Computer-assisted image analysis was performed with a Leica DM500 microscope (Leica Microsystems, Wetzlar, Germany), Leica ICC50 W camera (Leica Microsystems, Wetzlar, Germany), and Image Pro Plus 7.0 (Media Cybernetics, Inc., Rockville, MD, USA) software. The expression of tight junction (TJ) proteins: ZO-1, occludin and claudin-1 was blindly assessed by choosing random five fields on each slide (n = 4/group). Barrier TJ protein expressions and staining intensity in colonic epithelium were expressed as the percentage expression of a respective TJ protein.

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