Hematoxylin and Eosin Staining to Assess Colon Damage in DSS-Induced Colitis

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.

For hematoxylin and eosin staining of the isolated colon tissue, the next morning, submerge the tissue pieces in 30% sucrose in PBS overnight in individual 15-milliliter tubes for cryo-protection of the samples. The next day, embed the tissues in optimal cutting temperature or OCT compound and cool the tissues at minus 20 degrees Celsius until the OCT hardens.

Place the OCT blocks in a cryostat and set the thickness dial to 12 micrometers, then, slice and collect 12-micrometer thick frozen sections onto glass microscope slides. When all of the tissues have been sectioned, warm the slides at 65 degrees Celsius on a hot plate for 20 minutes, and wash the heated sections briefly in distilled water before staining with hematoxylin staining solution for 5 minutes.

Remove the excess hematoxylin solution in running tap water for 5 minutes, and differentiate the sections with 0.5% hydrochloric acid in ethanol for 30 seconds, followed by a 1-minute rinse under running tap water. Next, wash the samples for 1 minute in PBS, followed by another 5-minute wash under running tap water.

At the end of the wash, submerge the tissues in an ascending ethanol series for 10 seconds per immersion, followed by counter-staining in eosin for 2 minutes. For dehydration of the samples, submerge the slides in 95% ethanol and 2 changes of 100% ethanol for 5 minutes per immersion, followed by clearing with two 5-minute changes in xylene. Then, score the tissue damage to the proximal, middle, and distal colons of each experimental group.