Name: visualization of estrogen receptors in colons of mice with tnbs-induced crohn's disease using immunofluorescence
Uploaded: 2020-03-12T12:30:00
Description: 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

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 ...

<ol>
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Disease development is prevented by cotransfer of purified CD4+ T cells.</a> Journal of Experimental Medicine. 178 (1), 237-244 (1993).</li><li>K&#252;hn, R., L&#246;hler, J., Rennick, D., Rajewsky, K., M&#252;ller, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-10-deficient+mice+develop+chronic+enterocolitis.">Interleukin-10-deficient mice develop chronic enterocolitis.</a> Cell. 75 (2), 263-274 (1993).</li><li>Sundberg, J. P., Elson, C. O., Bedigian, H., Birkenmeier, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous,+heritable+colitis+in+a+new+substrain+of+C3H/HeJ+mice.">Spontaneous, heritable colitis in a new substrain of C3H/HeJ mice.</a> Gastroenterology. 107 (6), 1726-1735 (1994).</li><li>Harnish, D. 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A preliminary study.</a> Journal of Gastrointestinal and Liver Diseases. 26 (1), 29-35 (2017).</li><li>Mohammad, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+&#945;+contributes+to+T+cell-mediated+autoimmune+inflammation+by+promoting+T+cell+activation+and+proliferation.">Estrogen receptor &#945; contributes to T cell-mediated autoimmune inflammation by promoting T cell activation and proliferation.</a> Science Signaling. 11 (526), eaap9415 (2018).</li><li>Jacenik, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-coupled+estrogen+receptor+mediates+anti-inflammatory+action+in+Crohn's+disease.">G protein-coupled estrogen receptor mediates anti-inflammatory action in Crohn's disease.</a> Scientific Reports. 9 (1), 6749 (2019).</li><li>Prossnitz, E. R., Barton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+G+protein-coupled+estrogen+receptor+GPER+in+health+and+disease.">The G protein-coupled estrogen receptor GPER in health and disease.</a> Nature Review Endocrinology. 7 (12), 715-726 (2011).</li><li>Ya&#351;ar, P., Ayaz, G., User, S. D., G&#252;p&#252;r, G., Muyan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+estrogen-estrogen+receptor+signaling.">Molecular mechanisms of estrogen-estrogen receptor signaling.</a> Reproductive Medicine and Biology. 16 (1), 4-20 (2016).</li><li>Pizarro, T. T., Arseneau, K. 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L., St&#252;ber, E., Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+interleukin+12+abrogate+established+experimental+colitis+in+mice.">Antibodies to interleukin 12 abrogate established experimental colitis in mice.</a> Journal of Experimental Medicine. 182 (5), 1281-1290 (1995).</li><li>Ikeda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simvastatin+attenuates+trinitrobenzene+sulfonic+acid-induced+colitis,+but+not+oxazalone-induced+colitis.">Simvastatin attenuates trinitrobenzene sulfonic acid-induced colitis, but not oxazalone-induced colitis.</a> Digestive Diseases and Sciences. 53 (7), 1869-1875 (2007).</li><li>Thavarajah, R., Mudimbaimannar, V. K., Elizabeth, J., Rao, U. 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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 ...

Increasing evidence suggests the impact of estrogen signaling on the colonic part of physiology, immunohistochemistry is a promising technique for identifying estrogen receptors in the colon and they evolve into colitis. We present a complete and validated protocol for immunohistochemical visualization of estrogen receptors in the colon, using immunofluorescence. Together with Dr.Sylwia Michlewska from Laboratory of Microscopic Imaging of the University of Lodz, we will demonstrate the procedure.Place the colon in a petri dish. Cut the colon into one to two centimeter fragments. Place each fragment on a sponge in an appropriately labeled histological cassette.Place a cassette containing a colon fragment in 4%paraformaldehyde. Incubate for at least 24 hours at four degrees celsius. Prepare and program the tissue processor for one hour of incubation in 50%70%90%95%and 100%ethanol, xylene 100%ethanol, and xylene only, as well as for at least three hours of incubation in liquid paraffin.Transfer the colon fragment to a histological box. Place the box in the pre-programmed tissue processor and run the processor. After incubation in the tissue processor remove the colon from the histological box and place the colon fragment in a metal mold, such that the two ends of the colon are in an upright position.Fill one third of the mold with liquid paraffin. Place the mold in the cooling area at minus five degrees celsius for a few seconds and then move the mold to the warming area at 70 degrees celsius. Then place the mold in the bottom part of the histological box and cover the entire colon fragment with liquid paraffin.Place the metal mold containing the colon fragment and paraffin in the cooling area for a few minutes. Then remove the metal mold from the colon and paraffin block. Incubate the colon and paraffin block for at least 24 hours at four degrees celsius.After incubation, remove excess paraffin from the block. Insert the colon and paraffin block into a fully automated rotary microtome. Cut the colon fragment into five micrometer sections.Transfer one colon section to a water bath, preheated to 40 degrees celsius. Use the labeled glass slide to remove the colon section from the water bath and incubate the glass slide at room temperature for 24 hours. First, remove the paraffin by incubating the glass slide in xylene for five minutes.Repeat this step three times. Then place the glass slide in a one-to-one mixture of xylene and 100%ethanol for five minutes. Repeat this step three times.Use a series of decreasing ethanol concentrations to rehydrate the colon section. Immerse the slide in the ethanol for five minutes. Repeat three times at each concentration before proceeding to the next concentration.Rinse the glass slide under running water for five minutes. Reheat the antigen retrieval buffer to 95 to 98 degrees celsius. Then heat the glass slide in boiling antigen retrieval solution for 10 minutes.To prevent waste of reagents, use a hydrophobic pen to draw a circle around the colon section. Then, incubate the slide for 10 minutes in a 3%solution of hydrogen peroxidase and water. Wash the slide in washing solution for five minutes.Then incubate the slide in blocking solution for one hour at room temperature. Remove the blocking solution and add 20 to 50 microliters of primary antibody against E-R alpha, E-R beta, or G-P-E-R diluted. Incubate the primary antibody overnight at four degrees celsius in darkness.Remove the antibody solution. Wash the slide three times in washing solution for five minutes each time. Next, add diluted secondary antibody.Incubate the slide with secondary antibody conjugated with dye for one hour at room temperature in darkness. Remove the secondary antibody solution from the slide. Wash the slide three times in washing solution for five minutes each time.Add diluted fluorochrome for membrane visualization and incubate for 10 minutes at room temperature in darkness. Remove the solution and wash the slide three times in washing solution for five minutes each time. Add a few drops of glycerol-based liquid DAPI, a fluorochrome for cell nuclei visualization, directly on the colon section.Cover it carefully with a cover slide. Incubate the colon section for at least 24 hours at four degrees celsius. Analyze the colon section under a confocal microscope featuring 20x or 63x objectives and oil immersion.The specificity of the antibodies used in the study was validated using MCF-7 cells. The estrogen receptor antibodies enabled detection of nuclear estrogen receptors E-R alpha and E-R beta as well as the membrane-bound estrogen receptor G-P-E-R. A negative control was also performed in which MCF-7 cells were stained only with secondary antibody conjugated with fluorochrome and a glycerol-based liquid with DAPI.A strong cytoplasmic signal of E-R alpha was found in the colon sections obtained from control mice and from mice with TNBS-induced Crohn's disease. However, only the colons obtained from control mice had E-R alpha localized in the goblet cell cytoplasm. Confocal microscopy also revealed cytoplasmic localization of E-R beta in the colon sections of both control and the TNBS-treated mice.Similarly, cytoplasmic localization of G-P-E-R was documented in the colon sections obtained from control mice and the TNBS-treated mice. The correct positioning of the colon in the mold is essential to generate the correct cross section. Fluorochrome should be selected by its own excitation and emission spectrum.This method can also be applied to other proteins that may be involved in the development of colitis. Immunohistochemistry detection by immunofluorescence can be extended to stain other proteins in protein-protein direction status.

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

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 ...

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 ...

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 ...

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 ...

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 ...

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

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 ...

Modeling an Enzyme Active Site using Molecular Visualization Freeware

Biomolecular visualization skills are paramount to understanding key concepts in the biological sciences, such as structure-function relationships and ...