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

Overview

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.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Mice and DSS

1. Keep the knockout (α-gustducin^-/-) mice and age-, gender-, and body weight-matched wild-type control (α-gustducin^+/+) C57BL/6 mice individually in clean cages.
   NOTE: The knockout mice have been backcrossed with C57BL/6 mice for over 20 generations and have a nearly 100% C57BL/6 genetic background.
2. Dissolve 30 g of dextran sulfate sodium (DSS) powder in 1 L of autoclaved water. Mix until the solution becomes clear to ensure that the final working concentration is 3% (w/v).
   NOTE: The DSS solution can be stored at room temperature for up to 1 week or at 4 °C until use.

2. Induction and Evaluation of DSS Colitis in Mice

1. Weigh and record each mouse's initial body weight. Place the mice individually into standard plastic cages and label the cages.
2. Replace regular drinking water with 3% DSS solution for a total of 7 days to which both groups of mice have access ad libitum.
3. Measure the mouse's body weight, record DSS solution consumption, and collect and examine the stool of each mouse daily during the DSS administration. Observe the severity of diarrhea and rectal bleeding and convert this to the DSS-induced disease index.
4. During the experiment, the percentage of weight loss compared to initial weight and the disease index are calculated to evaluate the symptoms of colitis.
   NOTE: The disease index is scored by combining observations of diarrhea and rectal bleeding and is defined as follows: 0 (normal stool, no blood), 1 (soft stool, no blood), 2 (soft stool, little blood), 3 (very soft stool, modest bleeding), and 4 (watery stool, significant bleeding). The disease index is analyzed daily for each mouse.
5. By the end of the 7-day DSS treatment, sacrifice the mice by cervical dislocation and proceed with the remaining experiments.

3. Preparation of Tissue Samples

1. Place the mouse in the supine position and clean the skin of the abdomen with 70% ethanol. Make a 3 cm-long midline incision in the abdomen with a pair of small scissors to expose the abdominal cavity.
2. Use a pair of forceps to carefully separate the spleen from other tissues, then remove the spleen and measure its size.
3. Identify and lift the colon with forceps and separate it from the surrounding mesentery. Pull out the whole colon until the cecum and rectum are visible.
4. Isolate the colon by transecting it at the colonocecal margin and deep in the pelvis to free the proximal and distal colon, respectively. Then, measure and record the length of the isolated colon. Be careful not to damage the colonic tissue during the dissecting procedure.
5. Flush the colon with 10 mL of ice-cold phosphate-buffered saline (PBS) with a 10 mL syringe equipped with a gavage needle to remove the feces and blood until the eluate is completely clear.
6. For histological identification, divide the tissue samples equally into three parts: proximal, middle, and distal. Then, fix the tissue with 4% paraformaldehyde (PFA) overnight at 4 °C.
7. For the analysis of cytokine expression, freeze the entire colon rapidly with liquid nitrogen and store it at -80 °C until use.

4. Histological Assessment of the Severity of DSS-induced Colitis

1. Immunohistochemistry
   1. After fixation, submerge the tissue in a solution of 30% sucrose in 1x PBS overnight in a 15 mL tube to cryoprotect the samples.
   2. Embed the tissue in optimal cutting temperature (OCT) compound and place it at -20 °C until the OCT hardens.
   3. Transfer the OCT block containing the tissue to a cryostat, set the thickness dial in the cryostat to 12 µm, and slice and collect 12 µm-thick frozen sections.
   4. Heat the collected tissue sections from a cryostat at 65 °C for 20 min on a hot plate. Wash the sections in 1x PBS 3 times, for 10 min each. Incubate the tissue sections in 3% hydrogen peroxide for 10 min to eliminate endogenous peroxidase. Wash the sections 3 more times. Block the tissues with blocking buffer (3% bovine serum albumin (BSA), 0.3% non-ionic detergent, 2% goat serum, 0.1% sodium azide in 1x PBS) at room temperature for 1 h or more.
   5. Replace the blocking buffer with a solution containing the following immune cell type-specific primary antibodies: CD45 for leukocytes, CD3 for T cells, B220 for B cells, CD11b for macrophages, and Ly6G for neutrophils. Incubate at 4 °C overnight.
   6. Remove the primary antibodies from tissue sections by aspiration. Wash the sections with 1x PBS 3 times, for 10 min each. Incubate the sections with biotinylated secondary antibody followed by incubation with the streptavidin-HRP complex at room temperature.
   7. Incubate the sections with 3,3'-diaminobenzidine (DAB) solution to develop a light- or dark-brown color and visualize the immunoreactive cells.
   8. Counterstain the sections with hematoxylin and 0.3% (v/v) diluted ammonia. Take bright-field images at 10X magnification under a microscope.
   9. Use an image processing program to identify and quantify the population of the marked immune cells in both the epithelium and the lamina propria (underneath the epithelium) by setting 2 masks: use the first mask in the color-cube-based feature to set a color detection threshold and measure the colored DAB-reactive areas; use the second mask to determine total areas of the epithelium and laminar propria in the examined section. Express the immunoreactive cell population as a ratio of the staining area of the infiltrated cells to the total area of the examined tissue.

Materials

Name	Company	Catalog Number	Comments
Antibody
CD45	BD Biosciences	550539
CD3	BD Biosciences	555273
B220	BD Biosciences	550286
CD11b	BD Biosciences	550282
Ly6G	BD Biosciences	551459
Reagent
Dextran Sulfate Sodium Salt (DSS)	MP Biomedicals	2160110
Streptavidin-HRP complex	BD Pharmingen	551011
Phosphate-Buffered Saline (PBS)	Sangon Biotech	B548117
BD 10 ml Syringe	BD Biosciences	309604
Instruments and equipment
Balance
Scissors
Forceps
Staining jars
Software
Imag-Pro Plus	Media Cybernetics, Inc.