Name: hematoxylin and eosin staining to assess colon damage in dss-induced colitis
Uploaded: 2023-08-31T09:44:30.000+00:00
Duration: 1 min 47 s
Description: Source: Du, Y., et al. Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model. J. Vis. Exp. ...

hematoxylin and eosin staining to assess colon damage in dss-induced colitis

Hematoxylin and Eosin Staining to Assess Colon Damage in 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.

hematoxylin-eosin-staining-to-assess-colon-damage-dss-induced

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

EoE Sub Articles

eoe sub articles

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
<ol>
	<li>Keep the knockout (&#945;-gustducin-/-) mice and age-, gender-, and body weight-matched wild-type control (&#945;-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.</li>
	<li>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 &#176;C until use.</li>
</ol>
2. Induction and Evaluation of DSS Colitis in Mice
<ol>
	<li>Weigh and record each mouse&#39;s initial body weight. Place the mice individually into standard plastic cages and label the cages.</li>
	<li>Replace regular drinking water with 3% DSS solution for a total of 7 days to which both groups of mice have access ad libitum.</li>
	<li>Measure the mouse&#39;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.</li>
	<li>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.</li>
	<li>By the end of the 7-day DSS treatment, sacrifice the mice by cervical dislocation and proceed with the remaining experiments.</li>
</ol>
3. Preparation of Tissue Samples
<ol>
	<li>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.</li>
	<li>Use a pair of forceps to carefully separate the spleen from other tissues, then remove the spleen and measure its size.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
</ol>
4. Histological Assessment of the Severity of DSS-induced Colitis
<ol>
	<li>Hematoxylin and eosin (H&#38;E) staining
	<ol>
		<li>After fixation, submerge the tissue in a solution of 30% sucrose in 1x PBS overnight in a 15 mL tube to cryoprotect the samples.</li>
		<li>Embed the tissue in optimal cutting temperature (OCT) compound and place it at -20 &#176;C until the OCT hardens.</li>
		<li>Transfer the OCT block containing the tissue to a cryostat, set the thickness dial in the cryostat to 12 &#181;m, and slice and collect 12 &#181;m-thick frozen sections.</li>
		<li>Heat the collected tissue sections from a cryostat at 65 &#176;C for 20 min on a hot plate.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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&#38;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.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dextran Sulfate Sodium Salt (DSS)</td>
			<td>MP Biomedicals</td>
			<td>2160110</td>
			<td></td>
		</tr>
		<tr>
			<td>H&#38;E Staining Kit</td>
			<td>BBI Life Sciences</td>
			<td>E607318</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Sangon Biotech</td>
			<td>B548117</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 10 ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Staining jars</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imag-Pro Plus</td>
			<td>Media Cybernetics, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Du, Y., et al. <a href="https://app.jove.com/v/58668">Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model.</a> J. Vis. Exp. (2018)
This video demonstrates the hematoxylin and eosin (H&#38;E) staining method to evaluate colon damage in a murine model of DSS-induced colitis &#8212; an inflammatory bowel disease. Upon isolating the colon from a DSS-treated mouse and obtaining sections of the tissue, H&#38;E staining of the sections helps identify the inflammation-mediated tissue damage.

Source: Du, Y., et al. Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model. J. Vis. Exp. ...

Dextran sulfate sodium, or DSS, induces colitis &#8212; an inflammatory bowel disease &#8212; 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 &#8212; a metal cation &#8212; 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 &#8212; an alkaline medium &#8212; 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 &#8212; acidic eosin solution &#8212; 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 &#8212; for better microscopic examination.
Under a microscope, the colon section shows aggravated tissue damage, indicating DSS-induced colitis.

231 조회수. DSS 유발 대장염에서 결장 손상을 평가하기 위한 Hematoxylin 및 Eosin 염색

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

내레이션 대본