Hematoxylin and Eosin Staining to Assess Colon Damage in DSS-Induced Colitis
Transcrição
Dextran sulfate sodium, or DSS, induces colitis — an inflammatory bowel disease — where intestinal epithelial barrier disruption leads to inflammation.
To assess tissue damage, take fixed colon tissue isolated from a murine model of DSS-induced colitis. Immerse the tissue in an embedding medium for easy sectioning, and freeze rapidly.
Using a cryo-microtome, obtain a thin tissue section and collect it on a glass slide.
Dip the slide in a solution containing oxidized Hematoxylin dye bound to mordant — a metal cation — imparting a positive charge to the dye. The dye-mordant complex binds to negatively-charged DNA, staining the nucleus reddish-purple.
Submerge the tissue in a differentiating acid-alcohol solution, removing non-specifically bound dye from the cytoplasm and extracellular matrix. Rinse the tissue in a bluing agent — an alkaline medium — imparting a blue color to the nuclei, enhancing the contrast.
Immerse the slide in increasing alcohol concentrations to dehydrate the tissue enhancing counterstain penetration.
Dip the tissue in the counterstain — acidic eosin solution — imparting pink color to the basic cytoplasmic proteins and extracellular matrix. Wash the slide with alcohol to remove excess eosin. Dip the tissue in xylene to make the tissue transparent — for better microscopic examination.
Under a microscope, the colon section shows aggravated tissue damage, indicating DSS-induced colitis.
