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

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Mice and DSS

1. Keep the knockout (α-gustducin^-/-) mice and age-, gender-, and body weight-matched wild-type control (α-gustducin^+/+) C57BL/6 mice individually in clean cages.
   NOTE: The knockout mice have been backcrossed with C57BL/6 mice for over 20 generations and have a nearly 100% C57BL/6 genetic background.
2. Dissolve 30 g of dextran sulfate sodium (DSS) powder in 1 L of autoclaved water. Mix until the solution becomes clear to ensure that the final working concentration is 3% (w/v).
   NOTE: The DSS solution can be stored at room temperature for up to 1 week or at 4 °C until use.

2. Induction and Evaluation of DSS Colitis in Mice

1. Weigh and record each mouse's initial body weight. Place the mice individually into standard plastic cages and label the cages.
2. Replace regular drinking water with 3% DSS solution for a total of 7 days to which both groups of mice have access ad libitum.
3. Measure the mouse's body weight, record DSS solution consumption, and collect and examine the stool of each mouse daily during the DSS administration. Observe the severity of diarrhea and rectal bleeding and convert this to the DSS-induced disease index.
4. During the experiment, the percentage of weight loss compared to initial weight and the disease index are calculated to evaluate the symptoms of colitis.
   NOTE: The disease index is scored by combining observations of diarrhea and rectal bleeding and is defined as follows: 0 (normal stool, no blood), 1 (soft stool, no blood), 2 (soft stool, little blood), 3 (very soft stool, modest bleeding), and 4 (watery stool, significant bleeding). The disease index is analyzed daily for each mouse.
5. By the end of the 7-day DSS treatment, sacrifice the mice by cervical dislocation and proceed with the remaining experiments.

3. Preparation of Tissue Samples

1. Place the mouse in the supine position and clean the skin of the abdomen with 70% ethanol. Make a 3 cm-long midline incision in the abdomen with a pair of small scissors to expose the abdominal cavity.
2. Use a pair of forceps to carefully separate the spleen from other tissues, then remove the spleen and measure its size.
3. Identify and lift the colon with forceps and separate it from the surrounding mesentery. Pull out the whole colon until the cecum and rectum are visible.
4. Isolate the colon by transecting it at the colonocecal margin and deep in the pelvis to free the proximal and distal colon, respectively. Then, measure and record the length of the isolated colon. Be careful not to damage the colonic tissue during the dissecting procedure.
5. Flush the colon with 10 mL of ice-cold phosphate-buffered saline (PBS) with a 10 mL syringe equipped with a gavage needle to remove the feces and blood until the eluate is completely clear.
6. For histological identification, divide the tissue samples equally into three parts: proximal, middle, and distal. Then, fix the tissue with 4% paraformaldehyde (PFA) overnight at 4 °C.

4. Histological Assessment of the Severity of DSS-induced Colitis

1. Hematoxylin and eosin (H&E) staining
   1. After fixation, submerge the tissue in a solution of 30% sucrose in 1x PBS overnight in a 15 mL tube to cryoprotect the samples.
   2. Embed the tissue in optimal cutting temperature (OCT) compound and place it at -20 °C until the OCT hardens.
   3. Transfer the OCT block containing the tissue to a cryostat, set the thickness dial in the cryostat to 12 µm, and slice and collect 12 µm-thick frozen sections.
   4. Heat the collected tissue sections from a cryostat at 65 °C for 20 min on a hot plate.
   5. Wash the sections briefly in distilled water. Stain them with hematoxylin staining solution for 5 min and subsequently rinse with running tap water for 5 min.
   6. Differentiate the sections with 0.5% hydrochloric acid-ethanol for 30 s and rinse them in running tap water for 1 min. Then, perform the wash in 1x PBS for 1 min and subsequently rinse with running tap water for 5 min.
   7. Perform washing of the tissue sections in 70%, 75%, 80%, and 95% alcohol, each for 10 s. Counterstain in eosin staining solution for 2 min.
   8. Perform dehydration through 95% alcohol and 2 changes of absolute alcohol for 5 min each time. Clear in 2 changes of xylene for 5 min.
   9. Score tissue damage of the proximal, middle, and distal colon of each mouse for both the gene knockout and wild-type groups based on the results of the above H&E staining.
      NOTE: The disease index is a combined score of epithelial damage and inflammation in the mucosa, submucosa, muscularis, and serosa regions, which is defined as follows: 0 (no tissue damage and inflammation), 1 (focal tissue damage and inflammation), 2 (patchy tissue damage and inflammation), and 3 (diffuse tissue damage and inflammation). Three scores per mouse for the proximal, middle, and distal parts of the colon are then summed to obtain a total score for each animal. The average scores for each group are then calculated.