This method can help answer key questions in the inflammatory biodisease field about ulcerative colitis and Crohn's disease. The main advantage of this technique is that it can be used to evaluate first anti and proinfammatory effects of signaling proteins. The implication of this technique extends the survey of gas in new advances and inflammation.
Though this method can provide insights into genetic effect on gut inflammation. It can also be applied to other factors such as diets and microbiome. Generally, individuals new to this semester will struggle because processing disease scar tissue is tricky.
Visual demonstration of this method is critical as dissection and the tissue preparation steps are difficult to learn because mystalia and the cell tissue structures may be altered. To induce dextran sulfate sodium, or DDS colitis, replace the regular drinking water with 3%DSS solution, add libidium for seven days. Measuring each mouses'body weight, DSS solution consumption, and stool production daily.
At the end of the seven day treatment, place the mouse in the supine position and use small scissors to make a three centimeter long midline incision in the abdomen. Harvest the spleen and measure its size. Then use forceps to lift the colon to allow it to be separated from the surrounding mesentery.
And extract the whole colon until the cecum and rectum are visible. Transect the tissue at the colonosecal margin. And deep within the pelvis to free the proximal and distal colon, respectively.
Measure and record the length of the isolated colon. And use a 10 milliliter syringe equipped with a gavage needle to flush the colon with 10 milliliters of ice cold PBS to remove the feces and blood until the illuate runs clear. For histological identification, divide the tissue samples into equally sized proximal, middle, and distal sections.
And fix the tissues in 4%paraformaldehyde overnight at 4 degrees Celsius. For hematoxylin and eosin staining of the isolated colon tissue, the next morning, submerge the tissue pieces in 30%sucrose and PBS overnight in individual 15 milliliter tubes for cryo protection of the samples. The next day, embedd the tissues in optimal cutting temperature or OCT compound.
And cool the tissues in minus 20 degrees Celsius until the OCT hardens. Place the OCT blocks in the cryostat and set the thickness dial to 12 micrometers. Then slice and collect 12 micrometer thick frozen sections onto glass microscope slides.
When all the tissues have been sectioned, warm the slides at 65 degrees Celsius on a hot plate for 20 minutes. And wash the heated sections briefly in distilled water before staining with hematoxylin staining solution for five minutes. Remove the excess hematoxylin solution and run in tap water for five minutes.
And differentiate the sections with 0.5%hydrochloric acid in ethanol for 30 seconds, followed by a one minute rinse under running tap water. Next, wash the samples for one minute in PBS. Followed by another five minute wash under running tap water.
At the end of the wash, submerge the tissues in an ascending ethanol series for 10 seconds per immersion. Followed by counterstaining in eosin for two minutes. For dehydration of the samples, submerge the slides in 95%ethanol and two changes of 100%ethanol for five minutes per immersion.
Followed by clearing with two, five minute changes in xylene. Then score the tissue damage to the proximal, middle, and distal colons of each experimental group. For immunohistochemical analysis, after warming the sections for 20 minutes on the hot plate, wash the slides three times in PBS for 10 minutes per wash.
And eliminate the endogenous peroxidase with a 10 minute 3%hydrogen peroxide incubation. At the end of the incubation, wash the samples three times in PBS as demonstrated and block any nonspecific binding with blocking buffer for at least one hour at room temperature. Then, replace the blocking buffer with the primary antibody cocktail of interest.
For an overnight incubation at 4 degrees Celsius. The next morning, aspirate the excess primary antibody solution and wash the slides three times in PBS. After the last wash, incubate the slides with the appropriate biotinylated secondary antibody cocktail.
Followed by labeling with streptavidin horseradish peroxidase complex at room temperature. To visualize the immunoreactive cells, label the samples with DAB until a light or dark brown color develops. And counterstain the sections with hematoxylin and 0.3%diluted ammonia.
When the slides have dried, use a light microscope to obtain bright field images of the tissue sections under a 10x magnification. And use an image processing program to identify and quantify the population of the marked immune cells in both the epithelium and the lamina propria. For gene expression analysis in DSS treated colons, extract RNA from minus 80 degrees Celsius thawed colonic tissue samples according to standard RNA extraction protocols.
And mix one microgram with the total RNA with 2.5 microliters of 20 millimolar oligo deoxythymidine 12 through 18 primers and one microliter of moloney murine leukemia virus reverse transcriptase. After a 60 minute incubation at 42 degrees Celsius, mix one microliter of the cDNA with 0.5%microliters of the appropriate forward and reverse quantitative PCR primers for the cytokines of interest and 2x fluorescent green dye. Then run the quantitative PCR under the appropriate parameters and calculate the relative gene expression according to standard protocols.
Compared to wildtype mice, alpha gustducin knockout mice exhibit a more severe colitis with excessive weight loss, diarrhea and intestinal bleeding. After seven days of DSS administration, histological analysis of the colon sections reveal a more aggravated tissue damage within the proximal, middle, and distal colons of the knockout mice, compared to their wildtype counterparts. This excessive immune activation leads to the induction of colitis with a robust infiltration of various inflammatory cells in the knockout animal colon as observed by immunohistochemical analysis.
Further, quantitative PCR analysis demonstrates higher levels of TNF alpha and inferon gamma expression but lower expression of IL-5, 10, and 13, in knockout compared to wildtype mouse colons with no difference in TGF beta one expression observed. While attempting this procedure, it's important to remember to optimize textorous southvater sodium dosage and demonstration duration. Forwarding this procedure, other method like obtaining culture can be performed in order to understand additional questions like the effect of inflammation in intestinal tissue origination.
After it's development, this technique paves a way for researchers in the field of gut inflammation to explore the contributions of gene mutations to the severity of biodisease erodings and other vertebrates. Don't forget that working with disease gut tissue and its contents can be extremely hazardous and the repercussions such as wearing things that are appropriate, personal protection permanent should always be taken while performing this procedure.
