The overall goal of this technique is to efficiently process long sections of mouse intestinal tissue for uniform histological staining along its length. To get the intestinal sections there can be variations in staining along the entire length of the intestine. I tried utilizing the standard Swiss rolling technique but found it yields intestinal tissue that is very stiff and not easy to open.

With some experimentation, I came up with a quick fixation method that uses less toxic chemicals and that allows much better Swiss rolling of the intestinal tissue. The modified fixative can be used to quickly fix other thick tissues. In preparation, euthanize the test animal according to the text protocol and ready the surgical setting.

To begin, use dissection forceps and scissors to make a lateral incision into the middle of the abdomen about one to two centimeters below the ribcage depending on the size of the mouse. Then gently pull apart and cut away the skin to expose the abdominal muscles. Next, grip and pull up on the muscles without holding or pulling at the intestines.

Then, carefully make an initial incision in the abdominal muscles and carefully cut through them to expose the intestines. Now identify the colon and trace it to its distal end where it joins the rectum. Then using scissors transect the colon as close to the anal opening as possible.

Next find where the stomach joins the small intestines and using scissors cut free the intestines about one centimeter from the stomach. Now transfer the entire length of the free small intestine and colon to a clean petri dish containing PBS. Next secure the distal end of the colon and gently unravel the entire length of the colon removing any attached mesenteric tissues.

Then identify the cecum, and transect the intestine where the cecum joins the colon, thus isolating the colon. Similarly unravel the rest of the small intestine from the attached mesenteric connective and fatty tissues. Then transect the small intestine where it joins the cecum.

The cecum may then be removed and discarded. Now fill a 10 milliliter syringe with modified Bouin's fixative and attach a gavage needle. Insert the needle about half a centimeter into the anterior opening of an intestinal segment.

While firmly securing the gavage needle inside the segment, slowly flush the contents of the tissue with fixative. Be careful, as too much pressure will burst the segment. If the small intestine looks too long to handle, you might want to cut it into three equal segments first and flush each segment separately.

The fixation will cause the intestinal segment's color to become opaque. Next using scissors cut the segment longitudinally along the mesenteric line and then briefly rinse the segment in a dish of PBS while holding it open with forceps. Now cut the small intestine into three equal segments, proximal, mid and distal, which correspond roughly to the duodenum, the jejunum and the ileum.

Macroscopically, there are no discerning features to each section. Be sure to make a mark to identify the proximal from the distal end on each segment. Then place a segment with the lumenal side facing upward on the lid of a petri dish.

On the colon segment, identify the lumenal side by the ridges running across its width at the proximal end. The mid and distal regions have some variegations that run longitudinally. On the small intestine, identify the lumenal side by the rougher surface which marks the villi.

Microscopic examination shows that the villi are longest in the proximal regions of each segment, whereas the colonic crypts are shortest in the proximal regions and are at their tallest in the mid section. Proceed with Swiss rolling of the colon. With the colon flat open and the lumenal side up, pull it with forceps from its proximal end toward the edge of the petri dish.

Keeping the lumenal side up, hold the proximal end with the forceps and wrap up the edge of the proximal end round a toothpick and tightly pinch the wrapped edge against the toothpick. Then gently and slowly roll the colon around the toothpick to form a Swiss roll. Once the entire colon length has been rolled up, use another pair of forceps to carefully slide the roll into a tissue processing cassette.

Then transfer the cassette into 10 percent buffered formalin and allow it to incubate overnight at room temperature. To Swiss roll a segment of small intestine, open it lumenal side up and pull it with forceps from its proximal end toward the edge of a dish. Then use the same technique as shown with the colon to make the Swiss roll and fix it in formalin.

Using the described methods, HND staining for the large bowel of a C57 black 6 mouse revealed all portions of the colon for a comprehensive histological assessment. Next, the preparation was used for mice expressing EGFP driven by the LGR5 promoter which is active only in peripherating intestinal crypts. This EGFP transgene only has about 5 to 10 percent penetrance, but regions of interest could be easily found.

Using the same mice with EGFP LGR5 expression, KLF5 and KI67 were stained. First control mice with normal LGR5 expression were studied. EGFP positive cells are marked with blue arrows and EGFP negative cells are marked by white arrows.

Then the same transgenes were expressed in a KLF5 knockout mouse. Co-staining of KLF5 and KI67 is missing in EGFP positive stem cells, but remains present in EGFP negative crypts. Clearly the quality of the preparation for multi fluoro force staining is very good.

Once mastered, this technique can be done in about 15 minutes per mouse if you are collecting both the small intestine and the colon. Don't forget that working with Bouin's fixative can be extremely hazardous to the eyes. Precautions such as proper eye protection should always be taken while performing this procedure.

Thank you for watching and good look with your experiments.