Quantitative Immunohistochemical Analysis of Colonic Tight Junction Proteins

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 (link)]. 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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Shinde T., Perera A.P., Vemuri R., Gondalia S.V., Beale D.J., Karpe A.V., Shastri S., Basheer W., Southam B., Eri R, & Stanley R. (2020). Synbiotic supplementation with prebiotic green banana resistant starch and probiotic Bacillus coagulans spores ameliorates gut inflammation in mouse model of inflammatory bowel diseases. European Journal of Nutrition, 59(8), 3669-3689.

Publication 2020

(ABC) Detection IHC kit NBP1-85046 NBP1-87402 Leica DM500 microscope Leica ICC50 W camera Image Pro Plus 7.0

Antibodies Biologicals Claudin 1 Colonic Epithelium Microscope Novus Occludin Rabbit Tight junction Tight junction protein

Corresponding Organization :

Other organizations : University of Tasmania, Swinburne University of Technology, CSIRO Land and Water

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Antibodies used for incubating the colonic sections overnight at 4 °C, including anti-ZO-1, anti-occludin, and anti-claudin-1

dependent variables

Expression of tight junction (TJ) proteins: ZO-1, occludin and claudin-1
Staining intensity of TJ proteins in colonic epithelium

control variables

Colonic sections
Leica DM500 microscope, Leica ICC50 W camera, and Image Pro Plus 7.0 software used for computer-assisted image analysis
Random selection of five fields on each slide (n = 4/group) for assessment of TJ protein expressions and staining intensity

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Use a Rabbit specific HRP/DAB (ABC) Detection IHC kit or a Rabbit specific HRP Detection IHC kit for immunohistochemical detection of tight junction proteins (Protocol 1, 5)

Incubate the colonic sections with primary antibodies against tight junction proteins (ZO-1, occludin, claudin-1/claudin-3, Muc2) overnight at 4°C (Protocols 1, 2, 3, 4, 5)

Perform computer-assisted image analysis using a Nikon Eclipse 80i microscope or a Leica DM500 microscope, and Image Pro Plus 7.0 software (Protocols 1, 2, 5)

Blindly assess the expression of tight junction proteins by choosing a random number of crypts or fields per slide (Protocols 1, 2, 3, 5)

Use heat-induced epitope retrieval in a sodium citrate buffer, pH 6, to expose the tight junction protein epitopes (Protocol 5)

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