Why is null hypothesis testing critical in immune cell quantification?

Null hypothesis testing in immune cell quantification ensures that observed differences in immune infiltration are statistically significant, supporting robust target validation and reducing false positives in early discovery.

How does independent variable isolation improve colitis model assessment?

Isolating variables such as chemical treatment or antibody specificity allows clear attribution of immune cell changes to experimental interventions, strengthening mechanistic insights and discovery pipeline decisions.

What do quantitative immune cell measurements enable in preclinical studies?

Quantitative measurements of immune cell populations enable objective comparison of treatment effects, facilitate biomarker development, and support data-driven advancement of therapeutic candidates.

Why are replication requirements important for immunohistochemical analysis?

Replication ensures that immune cell quantification is reproducible across experiments and teams, enabling cross-functional collaboration and confidence in preclinical findings.

What statistical analysis capabilities are needed before implementing immune cell quantification?

Robust statistical analysis is required to validate differences in immune cell counts, assess variability, and confirm the reliability of immunohistochemical outputs for decision-making in R&D workflows.

Assessment of Chemical-Induced Colitis via Immunohistochemical Analysis of Colon Tissue Sections

JoVE Encyclopedia of Experiments

Immunology

0 views • 3:23 min • July 8th, 2025

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In mice, the colon mucosal layer consists of immune cells containing lamina propria, surrounded by a tightly packed epithelium that protects the mucosa. Chemical treatment with dextran sodium sulfate disrupts tight junctions, causing epithelial cell damage and enabling luminal microbes to penetrate the lamina propria.

Microbial recognition in the colon triggers chemokine secretion, leading to the infiltration of immune cells and the release of pro-inflammatory cytokines. This exaggerated immune response results in colon inflammation or colitis.

To assess chemical-induced colitis, begin with a glass slide carrying a fixed, pre-treated mouse colon tissue section exhibiting chemical-induced colitis. Treat the section with a cocktail of primary antibodies specific to the receptors of immune cells which bind to their respective immune cells.

Wash to remove unbound antibodies. Incubate the tissue section with biotinylated secondary antibodies, which bind specifically to the primary antibodies. Incubate with the streptavidin-enzyme conjugates, which interact with biotins, forming complexes.

Overlay the tissue section with a chromogenic substrate; the enzyme-substrate interaction imparts brown coloration to the immune cells. Counterstain the section with hematoxylin dye to stain the cell nuclei.

Observe the stained slide under a microscope. The abundance of brown immune cells in the damaged area indicates chemical-induced colitis.

For immunohistochemical analysis, after warming the sections for 20 minutes on the hot plate, wash the slides three times in PBS for 10 minutes per wash, and eliminate the endogenous peroxidase with a 10-minute 3% hydrogen peroxide incubation.

At the end of the incubation, wash the samples three times in PBS as demonstrated, and block any nonspecific binding with blocking buffer, for at least one hour at room temperature. Then, replace the blocking buffer with the primary antibody cocktail of interest for an overnight incubation at 4 degrees Celsius.

The next morning, aspirate the excess primary antibody solution, and wash the slides three times in PBS. After the last wash, incubate the slides with the appropriate biotinylated secondary antibody cocktail followed by labeling with streptavidin horseradish peroxidase complex at room temperature.

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Last updated: 15 August 2026

Assessment of Chemical-Induced Colitis via Immunohistochemical Analysis of Colon Tissue Sections

Overview

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Frequently Asked Questions

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Early Discovery & Target Validation

Screening & Assay Development

Translational & Preclinical Research

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