The work was published thanks to the financial support of the University of Lodz authorities: Vice-Rector for Scientific Research, Vice-Rector for National and International Cooperation and the Dean of the Faculty of Biology and Environmental Protection. Damian Jacenik was supported by grants (2017/24/T/NZ5/00045 and 2015/17/N/NZ5/00336) from National Science Centre, Poland.

Animal studies were conducted with the consent of the Local Ethical Committee (28/&#321;B29/2016) in accordance with Directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010, and institutional recommendations.
1. TNBS-induced murine model of Crohn&#39;s disease
NOTE: This protocol uses male BALB/C mice weighing 25-28 g. Animals are housed at a constant temperature (22-24 &#176;C) and, relative humidity 55 &#177; 5%, and maintained in a 12 h light/dark cycle with free access to standard chow pellets and tap water ad libitum.
<ol>
	<li>Place the mouse into the induction chamber and close the lid tightly. Anesthetize the mouse briefly with isoflurane (25% O2 with O2 flow rate at 1.5-2 L/min). 
	NOTE: Respiratory rate should remain rhythmic and slower than normal and should not change in response to a noxious stimulus.</li>
	<li>Instill 4 mg of TNBS in 0.1 mL of 30% ethanol in 0.9% NaCl or 0.1 mL of 30% ethanol in 0.9% NaCl as a vehicle control into the distal colon through a catheter. 
	NOTE: The catheter should be carefully introduced approximately 3 cm into the anus.</li>
	<li>Monitor the mouse daily from day two to eight for clinical parameters including body weight, rectal bleeding, stool consistency and mortality.</li>
	<li>On day eight, euthanize the mouse by cervical dislocation.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60813/60813fig1.jpg" /> 
Figure 1: Timeline for TNBS-induced murine model of Crohn&#39;s disease. <a href="https://www.jove.com/files/ftp_upload/60813/60813fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Separation and macroscopic evaluation of colon
NOTE: One day before colon separation, dilute 100 &#181;L of antibiotic in 1 mL of phosphate buffer saline (PBS) and leave at 4 &#176;C overnight.
<ol>
	<li>Clean the skin over the abdomen using 75% ethanol and sterile gauze.</li>
	<li>Cut the abdominal wall from breastbone to anus using sterile scissors and tweezers.</li>
	<li>Cut off the colon as close as possible to the anus and cecum.</li>
	<li>Place the colon on the Petri dish. Cut the colon along from the anus into the cecum end. Clean and wash the colon 2-4 times in cold antibiotic-PBS solution.</li>
	<li>Perform macroscopic evaluation using a caliper according to Table 1. 
	NOTE: Tissue adhesion* and erythema/hemorrhage#, fecal blood# and diarrhea# are subject to visual assessment. *Tissue adhesion evaluate using a three-point scale (0: colon without tissue adhesion, 1: colon with moderate tissue adhesion, 2: colon with extensive tissue adhesion); #based on absence (0) or presence (1) of erythema/hemorrhage, fecal blood and diarrhea.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesion*</td>
			<td>Erythema/ hemorrhage#</td>
			<td>Fecal blood#</td>
			<td>Diarrhea#</td>
			<td>Length of ulcer</td>
			<td>Colon thickness</td>
			<td>Colon length</td>
		</tr>
		<tr>
			<td>points (0 &#8211; 2)</td>
			<td>points (0 &#8211; 1)</td>
			<td>points (0 &#8211; 1)</td>
			<td>points (0 &#8211; 1)</td>
			<td>cm/points</td>
			<td>mm/points</td>
			<td>cm/points</td>
		</tr>
		<tr>
			<td>0 &#8211; absent</td>
			<td>0 &#8211; absent</td>
			<td>0 &#8211; absent</td>
			<td>0 &#8211; absent</td>
			<td rowspan="3">0.5 cm = 0.5 point</td>
			<td rowspan="3">n mm = n points</td>
			<td>0 &#8211; &#60;10% shorter than the control</td>
		</tr>
		<tr>
			<td>1 &#8211; moderate</td>
			<td>1 &#8211; present</td>
			<td>1 &#8211; present</td>
			<td>0.5 &#8211; slight/loose stool</td>
			<td>1 &#8211; from 10 to 20% shorter than the control</td>
		</tr>
		<tr>
			<td>2 &#8211; present</td>
			<td></td>
			<td></td>
			<td>1 &#8211; present</td>
			<td>2 &#8211; over 20% shorter than the control</td>
		</tr>
	</tbody>
</table>
Table 1: Macroscopic scoring of the intestine of mice with TNBS-induced model of Crohn&#39;s disease.
<ol start="6">
	<li>Convert the length of the ulcer in centimeters to a point scale, i.e., every 0.5 cm of ulcer is counted as 0.5 point. Convert the thickness of the colon in millimeters to a point scale, i.e., every n mm corresponds to n points.</li>
	<li>Convert the length of the colon in centimeters on a three-point scale. The length of colon obtained from each mouse with TNBS-induced Crohn&#39;s disease is evaluated in relation to the average colon length for the control group (0: &#60;10% shorter than the control, 1: from 10 to 20% shorter than the control, 2: over 20% shorter then the control).</li>
	<li>Calculate the total macroscopic score according to the equation: Total macroscopic score = adhesion (points) + erythema/hemorrhage (points) + fecal blood (points) + diarrhea (points) + length of ulcer (points) + colon thickness (points) + colon length (points).</li>
</ol>
3. Colon sample preparation
<ol>
	<li>Cut the colon into 1-2 cm fragments and place each on sponge in an appropriately labeled histological cassette. 
	NOTE: Sponges for histological cassettes prevent colon folding during dehydration and incubation in liquid paraffin.</li>
	<li>Place the colon fragment in 4% formaldehyde and incubate for at least 24 h at 4 &#176;C.</li>
	<li>Prepare and program the tissue processor for 1 h of incubation in 50%, 70%, 90%, 95%, 100% ethanol, xylene/100% ethanol (1:1; v/v), and xylene only, as well as for at least 3 h of incubation in liquid paraffin. 
	NOTE: Dehydration must be performed in increasing concentrations of ethanol and xylene, but the concentration of ethanol can be modified. The xylene/ethanol mixture is recommended but not required.</li>
	<li>Transfer the colon fragment to a histological box and place in the pre-programmed tissue processor.</li>
	<li>Run the tissue processor.</li>
	<li>After incubation steps, place the colon fragment in a metal mold so that the two ends of the colon are in an upright position and fill one third of the mold with liquid paraffin.</li>
	<li>Place the mold in the cooling area (-5 &#176;C) for a few seconds, and then move the mold to the warming area (70 &#176;C). Place in the bottom part of the histological box and cover the entire colon fragment with liquid paraffin.</li>
	<li>Leave the metal mold with the colon fragment in paraffin for a few minutes in the cooling area. Remove the metal mold from the paraffin block and incubate for at least 24 h at 4 &#176;C.</li>
	<li>Remove excess paraffin from the block and insert it into a fully automated rotary microtome. 
	NOTE: The paraffin block may be stored at -20 &#176;C for a few minutes before this step.</li>
	<li>Cut the colon fragment into 5 &#181;m sections.</li>
	<li>Transfer the colon section to a water bath preheated to 40 &#176;C.</li>
	<li>Use the labeled glass slide to remove the colon section from the water bath. 
	NOTE: The colon sections float on the water. Put the labeled glass slide in the water under the colon section and withdraw the glass slide carefully.</li>
	<li>Leave the glass slide for 24 h at room temperature. For long term storage, keep the glass slide at 4 &#176;C after 24 h of incubation at room temperature.</li>
</ol>
4. Immunohistochemistry with immunofluorescence staining
NOTE: Do not allow the colon section to dry at any step during the procedure.
<ol>
	<li>Remove paraffin by incubating the glass slide in xylene for 5 min. Repeat this step three times.</li>
	<li>Place the glass slide in xylene/100% ethanol (1:1; v/v) for 5 min. Repeat this step three times.</li>
	<li>Rehydrate the colon section in a series of decreasing ethanol concentrations, i.e., 70%, 50%, 30% and 10% ethanol for 5 min. Repeat each step three times.</li>
	<li>Rinse the glass slide under running water for 5 min.</li>
	<li>Preheat antigen retrieval buffer (10 mM sodium citrate; 0.05% Tween 20, pH 6.0) to 95-98 &#176;C and heat the glass slide in boiling antigen retrieval solution for 10 min. 
	NOTE: The antigen retrieval step is optional but recommended. The unmasking solution should be optimized depending on the antibody used in the experiment.</li>
	<li>Draw a circle around the colon section using a hydrophobic pen. 
	NOTE: This step is optional but recommended. The hydrophobic pen prevents waste of reagents by keeping the liquid pooled in a small volume inside marked the circle.</li>
	<li>Incubate the section in 3% water solution of hydrogen peroxidase for 10 min.</li>
	<li>Wash in washing solution (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.05% Tween 20) for 5 min.</li>
	<li>Incubate in blocking solution (5% normal goat serum; 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.05% Triton X-100) for 1 h at room temperature. 
	NOTE: In the blocking solution, the normal serum must be from the same species as the secondary antibody. In stages where incubation is required, place the glass slide in a humidity chamber to prevent excessive evaporation.</li>
	<li>Remove the blocking solution and add 20-50 &#181;L of primary antibody against ER&#945;, ER&#946; or GPER diluted in 1% bovine serum albumin with 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Triton X-100. 
	NOTE: Recommended dilutions of primary antibodies are shown in Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody type</td>
			<td>Antibody against</td>
			<td>Clonality</td>
			<td>Host species</td>
			<td>Species reactivity</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td rowspan="13">Primary</td>
			<td rowspan="4">ER&#945;</td>
			<td rowspan="4">Polyclonal</td>
			<td rowspan="4">Rabbit</td>
			<td>Human</td>
			<td rowspan="13">1:100</td>
		</tr>
		<tr>
			<td>Mouse</td>
		</tr>
		<tr>
			<td>Turtle</td>
		</tr>
		<tr>
			<td>Capybara</td>
		</tr>
		<tr>
			<td rowspan="6">ER&#946;</td>
			<td rowspan="6">Polyclonal</td>
			<td rowspan="6">Rabbit</td>
			<td>Human</td>
		</tr>
		<tr>
			<td>Monkey</td>
		</tr>
		<tr>
			<td>Rat</td>
		</tr>
		<tr>
			<td>Mouse</td>
		</tr>
		<tr>
			<td>Sheep</td>
		</tr>
		<tr>
			<td>Pig</td>
		</tr>
		<tr>
			<td rowspan="3">GPER</td>
			<td rowspan="3">Polyclonal</td>
			<td rowspan="3">Rabbit</td>
			<td>Human</td>
		</tr>
		<tr>
			<td>Rat</td>
		</tr>
		<tr>
			<td>Mouse</td>
		</tr>
		<tr>
			<td>Secondary</td>
			<td>DyLight 650</td>
			<td>Polyclonal</td>
			<td>Goat</td>
			<td>Rabbit</td>
			<td>1:250</td>
		</tr>
	</tbody>
</table>
Table 2: Characteristics of antibodies.
<ol start="11">
	<li>Incubate with primary antibody overnight at 4 &#176;C in darkness.</li>
	<li>Remove the antibody solution and wash in washing solution (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.05% Tween 20) for 5 min. Repeat this step three times.</li>
	<li>Add 20-50 &#181;L of DyLight 650 secondary antibody diluted in 1% bovine serum albumin (containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Triton X-100). Incubate with secondary antibody conjugated with dye for 1 h at room temperature in darkness. 
	NOTE: The recommended dilution of the secondary antibody is shown in Table 2.</li>
	<li>Remove the antibody solution and wash in washing solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 5 min. Repeat this step three times.</li>
	<li>Add 2% DiOC6(3) diluted in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl and incubate for 10 min at room temperature in darkness.</li>
	<li>Remove the solution and wash in washing solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 5 min. Repeat this step three times.</li>
	<li>Add a few drops of glycerol-based liquid with DAPI directly on the colon section and cover carefully with a cover slide. Incubate the colon section for least 24 h at 4 &#176;C. 
	NOTE: Avoid air bubbles when covering the tissue with the cover slide.</li>
	<li>Analyze the colon section under confocal microscope featuring 20x or 63x objectives and oil immersion using dedicated software. 
	NOTE: Table 3 lists characteristics of the fluorochromes used in this study.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Fluorochome type</td>
			<td colspan="2">Wavelength (nm)</td>
			<td rowspan="2">Dye</td>
		</tr>
		<tr>
			<td>Excitation</td>
			<td>Emission</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>405</td>
			<td>460 &#8211; 480</td>
			<td>Blue</td>
		</tr>
		<tr>
			<td>DiOC6 (3)</td>
			<td>485</td>
			<td>538 &#8211; 595</td>
			<td>Green</td>
		</tr>
		<tr>
			<td>DyLight 650</td>
			<td>654</td>
			<td>660 &#8211; 680</td>
			<td>Red</td>
		</tr>
	</tbody>
</table>
Table 3: Characteristics of fluorochromes.

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The authors have nothing to disclose.

There are numerous animal models for IBD pathophysiology examination, including genetic, immunological or spontaneous models, as well as chemically induced models15. Among the several types of animal models of colitis, chemically induced models such as the TNBS-induced model described in this protocol, are relatively inexpensive and easy to obtain. The TNBS-induced murine model of colitis has several clinical symptoms related to the pathological basis of CD. Animals with induced colitis are charac...

Crohn&#39;s disease (CD) is an inflammatory bowel disease (IBD) manifested as chronic intestine inflammation. The etiology of CD is poorly understood, but there are a few major factors that appear to be responsible for CD development, including intestinal microbiota, and genetic and environmental factors, such as diet or stress1. For a better understanding of the pathogenesis of Crohn&#39;s disease, several models of intestinal inflammation have been used2,3,4,5,6,7. In this article, we present results obtained from a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced murine model of CD.
It has been documented that estrogens are capable of modulating chronic intestinal inflammation8,9,10,11,12. The biological activity of estrogens is mediated by cognate receptors, among which are nuclear estrogen receptors (ERs), i.e., ER&#945; (gene ESR1) and ER&#946; (gene ESR2), as well as G protein-coupled estrogen receptor, i.e., GPER (gene GPER1), referred to as membrane-bound ER13,14. There are several methods for determining the level of estrogen receptors, but only a few can be used to visualize them in the intestine.
Immunohistochemistry (IHC) is a widely used method in clinical and basic studies for the detection of certain antigens in cells or tissues with fluorochrome-conjugated antibodies. IHC seems to be an important method in tissue structure visualization, as well as in the identification and localization of specific proteins, which may be crucial for understanding the development of colitis. Here, we present a complete and validated protocol for immunohistochemical visualization of estrogen receptors in the intestine using immunofluorescence.

<table><tbody><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BALB/C mice</td><td>University of Lodz</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Caliper</td><td>VWR</td><td>62379-531</td><td></td></tr><tr><td>Cardboard block</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Confocal microscope - TCS SP8</td><td>Leica Biosystems</td><td>NA</td><td></td></tr><tr><td>Fully automated rotary microtome - RM2255</td><td>Leica Biosystems</td><td>NA</td><td></td></tr><tr><td>Glass slide</td><td>Thermo Scientific</td><td>J1800BMNT</td><td></td></tr><tr><td>Heated Paraffin Embedding Module - EG1150 H</td><td>Leica Biosystems</td><td>NA</td><td></td></tr><tr><td>Histological box</td><td>Marfour</td><td>LN.138747</td><td></td></tr><tr><td>Hydrophobic pen</td><td>Sigma-Aldrich</td><td>Z377821</td><td></td></tr><tr><td>Laboratory balance</td><td>Radwag</td><td>WL-104-0048</td><td></td></tr><tr><td>LAS X software</td><td>Leica Biosystems</td><td>NA</td><td></td></tr><tr><td>Metal mold</td><td>Marfour</td><td>CP.5105</td><td></td></tr><tr><td>Sterile gauze</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Sterile scissor</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Sterile tweezer</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Tissue processor - TP1020</td><td>Leica Biosystems</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2, 4, 6-trinitrobenzene sulfonic acid</td><td>Sigma-Aldrich</td><td>92822</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A3294</td><td></td></tr><tr><td>DiOC6 (3)</td><td>Sigma-Aldrich</td><td>318426</td><td></td></tr><tr><td>DyLight 650 secondary antibody</td><td>Abcam</td><td>ab96886</td><td></td></tr><tr><td>ER&amp;alpha; primary antibody</td><td>Abcam</td><td>ab75635</td><td></td></tr><tr><td>ER&amp;beta; primary antibody</td><td>Abcam</td><td>ab3576</td><td></td></tr><tr><td>Ethanol</td><td>Avantor Performance Materials Poland</td><td>396480111</td><td></td></tr><tr><td>Formaldehyde</td><td>Avantor Performance Materials Poland</td><td>432173111</td><td></td></tr><tr><td>GPER primary antibody</td><td>Abcam</td><td>ab39742</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Avantor Performance Materials Poland</td><td>575283421</td><td></td></tr><tr><td>Hydrogen peroxidase</td><td>Avantor Performance Materials Poland</td><td>885193111</td><td></td></tr><tr><td>isoflurane (forane)</td><td>Baxter</td><td>1001936040</td><td></td></tr><tr><td>Normal goat serum</td><td>Gibco</td><td>16210064</td><td></td></tr><tr><td>Paraffin</td><td>Leica Biosystems</td><td>39602012</td><td></td></tr><tr><td>Petrie dish</td><td>Nest Scientific</td><td>705001</td><td></td></tr><tr><td>Phosphate buffer saline</td><td>Sigma-Aldrich</td><td>P3813</td><td></td></tr><tr><td>Physiological saline</td><td>Sigma-Aldrich</td><td>7982</td><td></td></tr><tr><td>Primocin (antibiotic)</td><td>Invitrogen</td><td>ant-pm-1</td><td></td></tr><tr><td>ProLong Diamond Antifage Mountant with DAPI (glycerol-based liquid with DAPI)</td><td>Invitrogen</td><td>P36971</td><td></td></tr><tr><td>Sodium chloride</td><td>Chempur</td><td>WE/231-598-3</td><td></td></tr><tr><td>Sodium citrate</td><td>Avantor Performance Materials Poland</td><td>795780429</td><td></td></tr><tr><td>Tris</td><td>Avantor Performance Materials Poland</td><td>853470115</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr><tr><td>Xylene</td><td>Avantor Performance Materials Poland</td><td>BA0860119</td><td></td></tr></tbody></table>

visualization of estrogen receptors in colons of mice with tnbs-induced crohn's disease using immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

Macroscopic characteristics of colons in mice with TNBS-induced Crohn&#39;s disease 
Representative images of colons taken from control and TNBS-treated mice are shown in Figure 2. In mice with a TNBS-induced model of Crohn&#39;s disease, the length of the colon is reduced while the width of the colon is increased.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/6081...

Watch this Scientific Journal Video about Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence at JoVE.com

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

visualization-estrogen-receptors-colons-mice-with-tnbs-induced-crohn

Research

JoVE Journal

Biochemistry

6.5K Views.  University of Lodz. Toenemende bewijs suggereert de impact van oestrogeen signalering op het colonische deel van de fysiologie, immunohistochemie is een veelbelovende techniek voor het identificeren van oestrogeenreceptoren in de dikke darm en ze evolueren in colitis. We presenteren een compleet en gevalideerd protocol voor immunohistochemische visualisatie van oestrogeenreceptoren in de dikke darm, met behulp van immunofluorescentie. Samen met Dr.Sylwia Michlewska van Laboratory of Microscopische Beeldvorming v...

Video: Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Cancer Research

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Medicine

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Immunology and Infection

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to ...

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis. Although anti-inflammatory therapies may help control inflammation, they have no efficacy on fibrosis and stenosis in CD. The pathogenesis of CD is not well understood. Current studies focus mainly on delineating dysregulated gut immune response mechanisms. While CD-associated transmural inflammation, intestinal fibrosis, and luminal stenosis all represent mechanical stress to the gut wall, the role of mechanical stress in CD is not well defined. To determine if mechanical stress plays an independent pathogenic role in CD, a protocol of TNBS-induced CD-like colitis model in rodents has been developed. This TNBS-induced transmural inflammation and fibrosis model resembles pathological hallmarks of CD in the colon. It is induced by intracolonic instillation of TNBS into the distal colon of adult Sprague-Dawley rats. In this model, transmural inflammation leads to stenosis at the TNBS instillation site (Site I). Mechanical distention is observed in the portion proximal to the instillation site (Site P), representing mechanical stress but not visible inflammation. Colonic portion distal to inflammation (Site D) presents neither inflammation nor mechanical stress. Distinctive changes of gene expression, immune response, fibrosis, and smooth muscle growth at different sites (P, I, and D) were observed, highlighting a profound impact of mechanical stress. Therefore, this model of CD-like colitis will help us better understand CD&#39;s pathogenic mechanisms, particularly the role of mechanical stress and mechanical stress-induced gene expression in immune dysregulation, intestinal fibrosis, and tissue remodeling in CD.

The present protocol describes the development of a Crohn's-like colitis model in rodents. Transmural inflammation leads to stenosis at the TNBS instillation site, and mechanical enlargement is observed in the segment proximal to the stenosis. These changes allow studying mechanical stress in colitis.

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Biology

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

Source: Du, Y. W. 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>&#160;J. Vis. Exp.&#160;(2018)
This video demonstrates the assessment of chemical-induced colitis via immunohistochemical staining of mouse colon tissue. In a stained tissue section, the presence of a large number of immune-responsive cells in the damaged tissue represents the infiltration of immune cells during colitis.

Source: Du, Y. W. et al., Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model.&#160;J. Vis. ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

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