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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animals</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>Equipment</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>Reagents</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&#945; primary antibody</td>
			<td>Abcam</td>
			<td>ab75635</td>
			<td></td>
		</tr>
		<tr>
			<td>ER&#946; 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. The protocol presents a complete validated TNBS-induced murine model of Crohn's disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

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

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy

Immunofluorescence is a common method used to visualize subcellular compartments and to determine the localization of specific proteins within a tissue sample. A great hindrance to the acquisition of high quality immunofluorescence images is endogenous autofluorescence of the tissue caused by aging pigments such as lipofuscin or by common sample preparation processes such as aldehyde fixation. This protocol describes how background fluorescence can be greatly reduced through photobleaching using white phosphor light emitting diode (LED) arrays prior to treatment with fluorescent probes. The broad-spectrum emission of white phosphor LEDs allow for bleaching of fluorophores across a range of emission peaks. The photobleaching apparatus can be constructed from off-the-shelf components at very low cost and offers an accessible alternative to commercially available chemical quenchers. A photobleaching pre-treatment of the tissue followed by conventional immunofluorescence staining generates images free of background autofluorescence. Compared to established chemical quenchers which reduced probe as well as background signals, photobleaching treatment had no effect on probe fluorescence intensity while it effectively reduced background and lipofuscin fluorescence. Although photobleaching requires more time for pre-treatment, higher intensity LED arrays may be used to reduce photobleaching time. This simple method can potentially be applied to a variety of tissues, particularly postmitotic tissues that accumulate lipofuscin such as the brain and cardiac or skeletal muscles.

Background autofluorescence of biological samples often complicates fluorescence-based imaging techniques, especially in aged human postmitotic tissues. This protocol describes how autofluorescence from these samples can be effectively removed using a commercially available light emitting diode light source to photobleach the sample prior to immunostaining.

Immunofluorescence is a common method used to visualize subcellular compartments and to determine the localization of specific proteins within a tissue ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled antibodies. It is a multistep process in which the optimization of each step is crucial to obtain the optimum specific signal. Through IHC, the distribution and localization of specific biomarkers can be detected, revealing information on evolutionary conservation. Moreover, IHC allows for the understanding of expression and distribution changes of biomarkers in pathological conditions, such as obesity. IHC, mainly the immunofluorescence technique, can be used in adult zebrafish to detect the organization and distribution of phylogenetically conserved molecules, but a standard IHC protocol is not estasblished. Orexin and endocannabinoid are two highly conserved systems involved in the control of food intake and obesity pathology. Reported here are protocols used to obtain information about orexin peptide (OXA), orexin receptor (OX-2R), and cannabinoid receptor (CB1R) localization and distribution in the gut and brain of normal and diet-induced obese (DIO) adult zebrafish models. Also described are methods for immunoperoxidase and double immunofluorescence, as well as preparation of reagents, fixation, paraffin-embedding, and cryoprotection of zebrafish tissue and preparation for an endogenous activity-blocking step and background counterstaining. The complete set of parameters is obtained from previous IHC experiments, through which we have shown how immunofluorescence can help with the understanding of OXs, OX-2R, and CB1R distribution, localization, and conservation of expression in adult zebrafish tissues. The resulting images with highly specific signal intensity led to the confirmation that zebrafish are suitable animal models for immunohistochemical studies of distribution, localization, and evolutionary conservation of specific biomarkers in physiological and pathological conditions. The protocols presented here are recommended for IHC experiments in adult zebrafish.

Presented here are protocols for immunohistochemical characterization and localization of orexin peptide, orexin receptors, and endocannabinoid receptors in the gut and brains of normal and diet-induced obesity (DIO) adult zebrafish models using immunoperixidase and double immunofluorescence methods.

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled ...

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study reveals that transplantation of neonatal cardiac macrophages recruited by injury promotes adult heart regeneration after myocardial infarction with improvement of cardiac function and cardiomyocyte proliferation. The results indicate that neonatal cardiac macrophage transplantation could be a promising strategy for cardiac injury treatment. Here, we provide the technical details, including the isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages into myocardial-infarcted adult mice, and the estimation of heart regeneration after a macrophage graft.

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study ...

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. 
The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.