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

Summary

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.

Abstract

Crohn'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's disease. A detailed protocol for the induction of Crohn'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.

Introduction

Crohn'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's disease, several models of intestinal inflammation have been used^2,3,4,5,6,

Protocol

Animal studies were conducted with the consent of the Local Ethical Committee (28/ŁB29/2016) 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's disease

NOTE: This protocol uses male BALB/C mice weighing 25-28 g. Animals are housed at a constant temperature (22-24 °C) and, relative humidity 55 ± 5%, and maintained in a 12 h light/dark cycle with free access to standard chow pellets and tap water ad libitum.

1. Place the mouse into the induction chamber and close the....

Results

Macroscopic characteristics of colons in mice with TNBS-induced Crohn'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's disease, the length of the colon is reduced while the width of the colon is increased.

Discussion

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.......

Materials

List of materials used in this article

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