This study investigates the effects of chemical treatment on mouse colon tissue, focusing on the disruption of tight junctions and subsequent immune response. The methodology includes immunohistochemical analysis to assess the presence of immune cells in the context of colitis.
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.
To visualize the immunoreactive cells, label the samples with DAB until a light or dark brown color develops and counterstain the sections with hematoxylin and 0.3% diluted ammonia.
When the slides have dried, use a light microscope to obtain brightfield images of the tissue sections under a 10 times magnification, and use an image processing program to identify and quantify the population of the marked immune cells in both the epithelium and the lamina propria.
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