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