Whole Mount Preparation of Mouse Intestine: Using a Simple Gut-incising Device to Prepare Whole Mount

Whole-mount analysis allows the visualization of multi-dimensional tissue cross-sections while preserving the structural interactions between cells and their microenvironment.

To prepare whole mounts of the intestine, begin with a freshly harvested mouse intestine. Remove the mesentery - folds of peritoneal tissue - from the intestine. Flush the intestine with a chilled buffer to remove any fecal matter.

Next, excise the cecum.  Dissect the intestine into equal segments. Make small incisions in the intestinal segments to drain the fluid within them.  

Gently insert a wet stainless-steel rod into the intestinal segments. Now, position these rods into holding slots at the base of the customized gut-incising device. Fit the lid of the device over its base.

Using angled bars on the lid as the guide, longitudinally incise the intestinal segments. Remove the lid from the device. Moisten the segments. Gently roll the rod sideways to release and spread the segments flat.

Immerse the segments in a bath containing a suitable fixative and incubate for the desired duration. The fixative chemically preserves the cellular structure and tissue architecture for subsequent sampling.

Following fixation, store the tissue in ethanol until further downstream analysis.

This procedure uses a device consisting of the base that has high elevated strips at each end with grooves to take the rods and the lid. After harvesting the small intestine and colon from an adult mouse, hold the mesentery with curved forceps and gently pull the tissue away from the intestine to remove it. Place a Gilson-type pipette tip, cut at the wider end, onto a 10- or 20-milliliter luer fitting plastic syringe, and rinse the intestines with cold PBS.

After the tissue has been washed, lay out the small bowel and colon on a paper towel, and use scissors to divide the small bowel into three equal sections. Make several very small cuts in the intestinal segments to drain the fluid. Then, place another paper towel over the intestines and gently run a finger over the segments to squeeze out the remaining fluid.

Next, blot the preparations dry and gently insert stainless steel rods soaked in PBS into the tissues. Using a pencil, create labels for the segments including the autopsy date, experimental code, and animal identification number. Place the labels in the base of the device. Then, insert the rods and intestines into the slots at the base of the device, taking care that the proximal end of the segments are positioned in a standardized way near the labels.

Now, place the top piece of the device over the base, and use the angled bars to guide a scalpel blade longitudinally through the segments. To keep the tissue in place during the sectioning, carefully hold the segments with a finger so that they do not move with the knife.

After all of the sections have been made, remove the top of the device and use a piece of stiff card to carefully remove the filter paper and the tissues. Using a gloved fingertip dipped in PBS, slightly wet the segments. Gently roll the rod, side to side, to open up the gut and to spread the tissue flat.

The sections will adhere to the filter paper. After visually examining the preparations for any gross lesions, transfer the filter paper to a shallow bath of fixative. When the tissues are fixed, transfer them into 70% ethanol for storage until further downstream analysis.