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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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^2,3,4,5,6,7. 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 inflammation^8,9,10,11,12. The biological activity of estrogens is mediated by cognate receptors, among which are nuclear estrogen receptors (ERs), i.e., ERα (gene ESR1) and ERβ (gene ESR2), as well as G protein-coupled estrogen receptor, i.e., GPER (gene GPER1), referred to as membrane-bound ER^13,14. 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.

Protokół

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 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.
2. 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.
3. Monitor the mouse daily from day two to eight for clinical parameters including body weight, rectal bleeding, stool consistency and mortality.
4. On day eight, euthanize the mouse by cervical dislocation.

Figure 1: Timeline for TNBS-induced murine model of Crohn's disease. Please click here to view a larger version of this figure.

2. Separation and macroscopic evaluation of colon

NOTE: One day before colon separation, dilute 100 µL of antibiotic in 1 mL of phosphate buffer saline (PBS) and leave at 4 °C overnight.

1. Clean the skin over the abdomen using 75% ethanol and sterile gauze.
2. Cut the abdominal wall from breastbone to anus using sterile scissors and tweezers.
3. Cut off the colon as close as possible to the anus and cecum.
4. 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.
5. 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.

Adhesion*	Erythema/ hemorrhage#	Fecal blood#	Diarrhea#	Length of ulcer	Colon thickness	Colon length
points (0 – 2)	points (0 – 1)	points (0 – 1)	points (0 – 1)	cm/points	mm/points	cm/points
0 – absent	0 – absent	0 – absent	0 – absent	0.5 cm = 0.5 point	n mm = n points	0 – <10% shorter than the control
1 – moderate	1 – present	1 – present	0.5 – slight/loose stool			1 – from 10 to 20% shorter than the control
2 – present			1 – present			2 – over 20% shorter than the control

Table 1: Macroscopic scoring of the intestine of mice with TNBS-induced model of Crohn's disease.

6. 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.
7. 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's disease is evaluated in relation to the average colon length for the control group (0: <10% shorter than the control, 1: from 10 to 20% shorter than the control, 2: over 20% shorter then the control).
8. 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).

3. Colon sample preparation

1. 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.
2. Place the colon fragment in 4% formaldehyde and incubate for at least 24 h at 4 °C.
3. 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.
4. Transfer the colon fragment to a histological box and place in the pre-programmed tissue processor.
5. Run the tissue processor.
6. 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.
7. Place the mold in the cooling area (-5 °C) for a few seconds, and then move the mold to the warming area (70 °C). Place in the bottom part of the histological box and cover the entire colon fragment with liquid paraffin.
8. 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 °C.
9. Remove excess paraffin from the block and insert it into a fully automated rotary microtome.
   NOTE: The paraffin block may be stored at -20 °C for a few minutes before this step.
10. Cut the colon fragment into 5 µm sections.
11. Transfer the colon section to a water bath preheated to 40 °C.
12. 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.
13. Leave the glass slide for 24 h at room temperature. For long term storage, keep the glass slide at 4 °C after 24 h of incubation at room temperature.

4. Immunohistochemistry with immunofluorescence staining

NOTE: Do not allow the colon section to dry at any step during the procedure.

1. Remove paraffin by incubating the glass slide in xylene for 5 min. Repeat this step three times.
2. Place the glass slide in xylene/100% ethanol (1:1; v/v) for 5 min. Repeat this step three times.
3. 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.
4. Rinse the glass slide under running water for 5 min.
5. Preheat antigen retrieval buffer (10 mM sodium citrate; 0.05% Tween 20, pH 6.0) to 95-98 °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.
6. 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.
7. Incubate the section in 3% water solution of hydrogen peroxidase for 10 min.
8. Wash in washing solution (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.05% Tween 20) for 5 min.
9. 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.
10. Remove the blocking solution and add 20-50 µL of primary antibody against ERα, ERβ 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.

Antibody type	Antibody against	Clonality	Host species	Species reactivity	Dilution
Primary	ERα	Polyclonal	Rabbit	Human	1:100
				Mouse
				Turtle
				Capybara
	ERβ	Polyclonal	Rabbit	Human
				Monkey
				Rat
				Mouse
				Sheep
				Pig
	GPER	Polyclonal	Rabbit	Human
				Rat
				Mouse
Secondary	DyLight 650	Polyclonal	Goat	Rabbit	1:250

Table 2: Characteristics of antibodies.

11. Incubate with primary antibody overnight at 4 °C in darkness.
12. 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.
13. Add 20-50 µL 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.
14. 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.
15. 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.
16. 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.
17. 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 °C.
    NOTE: Avoid air bubbles when covering the tissue with the cover slide.
18. 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.

Fluorochome type	Wavelength (nm)		Dye
	Excitation	Emission
DAPI	405	460 – 480	Blue
DiOC6 (3)	485	538 – 595	Green
DyLight 650	654	660 – 680	Red

Table 3: Characteristics of fluorochromes.

Wyniki

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.

Dyskusje

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...

Materiały

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