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

Overview

In this video, we demonstrate whole mount preparation of the mouse intestine using a simple gut-incising device. The resulting mounts are of superior and uniform quality and can be used for histological analyses.

Protocol

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

1. Device

1. Machine the main portion of the base of the device from out of a solid block of high-density acetyl resin, as described in this manuscript. At each end, create 3 mm high-elevated strips and part machine out of these 2.5 mm grooves to take the rods (see Figure 1 and Figure 2).
2. Make rods from a 2.4 mm diameter stainless steel with rounded-off ends.
NOTE:The main frame of the lid was machined out of Duralumin plate.

2. Use of the Device

1. Dissect the small intestine and colon from the abdominal cavity using scissors and gloved fingers. Remove the mesentery by holding it with curved forceps and gently pulling it away from the intestine.
2. Rinse the intestines with cold phosphate-buffered saline (PBS, pH 7.4) using a Gilson-type pipette tip, which had been cut down at its wider end so that it can fit onto a 10 or 20 mL luer fitting plastic syringe.
3. Lay out the small bowel (SB) onto a sheet of paper towel and divide into three equal sections in length (proximal, mid, and distal – SB1, SB2, and SB3) using scissors. Remove the cecum and lay out the colon.
4. Make several very small cuts into the intestinal segments with scissors to allow the fluid to escape and place a sheet of paper towel over the intestines and gently run a finger over the preparations to squeeze out the fluid and blot dry.
5. Lift the sections and insert the stainless-steel rods that were previously wetted by soaking in a beaker of phosphate-buffered saline.
   NOTE: The intestine can be inverted if so desired (turned inside out).
6. Label a piece of card or filter paper cut to size to fit into the base of the device. Pencil is best, as it is unaffected by virtually all solutions. Autopsy date, experimental code, and animal identification number are suggested.
7. Place the card or filter paper into the base of the device. Insert the rods and intestines into the slots in the base of the device. Make sure the proximal end of the segments is positioned in a standardized manner. The proximal ends should be near the card labels.
8. Place the top piece of the device over the base. Use the angled bars to guide a scalpel blade to cut the sections longitudinally.
   NOTE: It helps if the tissue is gently and carefully held with a finger to ensure that it does not move with the knife.
9. Remove the top piece of the device. Carefully remove the filter paper and tissues (still on their rods).
   NOTE: A piece of stiff card placed underneath the filter paper will aid lifting.
10. Slightly wet (with a gloved fingertip dipped in PBS) the segments. Roll the rod, side to side, to open up the gut and spread it flat. The tissue will then adhere to the filter paper.
11. Visually examine the preparations for any gross lesions.
12. Transfer the tissue adhered to the filter paper to a shallow bath (or sandwich box) containing a fixative.
    NOTE: After the tissue adheres to the filter paper or to the card, it will remain firmly attached. A wide range of fixatives can be used to preserve the preparation. It is recommended that Carnoy’s fluid should be used for the study of intestinal cell proliferation, which is the ideal fixation for the best cell proliferation methods; however, formalin or many other fixatives can also be used if other endpoints are required.
13. After fixation (usually 3 h), transfer the preparations into 70% ethanol. The samples can then be stored until required. The preparations can be studied en face and/or used for the preparation of histological samples for H&E or other histological staining techniques, immunohistochemistry, and/or in situ hybridization.
    NOTE: Prolonged fixation can reduce immunohistochemical reactivity and/or in situ hybridization reactivity. Polypropylene sandwich boxes are ideal for storing a large number of preparations.
14. If required, the fixed tissues from the various intestinal segments can be made into ‘Swiss rolls’ and stained with the entire range of histological methods, including those described above. To make a ‘Swiss roll’ the intestinal segments are removed from the filter paper, rolled around a cocktail stick or a toothpick, secured with an entomological pin, and processed for wax embedding.
15. If required, stain the bulk tissue with methylene blue to visualize aberrant crypt foci.
16. Score the fixed preparations at leisure.

Representative Results

Figure 1. Photos of the original device. The original device was constructed from several pieces of metal, prepared by hand. (A) The device assembled. (B) The top of the device has been removed to show the rods in position.

Figure 2. Photos of the redesigned device. (A) The main frame of the lid is machined out of Duralumin plate. (B) The base (seen in black) is made of a solid block of high-density acetyl resin, having 4 removable rods sitting in a channel. Shown is a filter paper (white) placed on the top of the base (black). (C) The final view of the device: the lid (A) is placed on the top of the basin (B). Dimensions in centimeters are shown. The rods were 2.4 mm in diameter with rounded-off ends. See reference 5 for more details.

Materials

Name	Company	Catalog Number	Comments
Delrin	Ryan Plastics, Earls Barton, UK		High-density acetal resin similar material would suffice
Duralumin plate	Metal Supplies Ltd, Park Road,Dukinfield. SK16 5LP UK		Aluminium allow dating back to 1909 so alterative suppliers are available
Finished device(s) ready for use	Contact Dr Goodlad  r.goodlad@imperial.ac.uk		Contact r.goodlad@imperial.ac.uk for supply details