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.
