TITLE: Whole mount dissection and staining of enteric nervous system
AUTHORS: Michael Henderson, Alice Prigent

[DESCRIPTION]
This protocol details whole mount dissection and staining of enteric nervous system.

[STEPS]
SECTION: Perfusion
1. Administer sodium pentobarbital through intraperitoneal injection.
SECTION: Perfusion
2. Place mouse back in the cage long enough for anesthesia to take effect.  Apply a hard toe pinch until mouse no longer reacts, ensuring that the mouse can no longer feel pain before proceeding.
SECTION: Perfusion
3. Place mouse, abdomen-up, on Styrofoam block. Spray mouse abdomen with 70% ethanol.  Grasp skin below ribcage with forceps and cut skin with scissors from middle up either side towards the armpits, cutting through ribcage. Diaphragm should carefully be cut circumferentially.
SECTION: Perfusion
4. Remove pericardium and peripheral fat to expose heart.
SECTION: Perfusion
5. Insert the needle into the left ventricle and secure it with vascular clamp. Make a small incision on the right atrium using fine scissors.
SECTION: Perfusion
6. Start the saline perfusion for 7 min at a constant speed of ~1 mL/10 s.
SECTION: Tissue collection
7. Following perfusion, open the abdomen of the mouse, remove and collect the stomach and duodenum.
SECTION: Tissue collection
8. Place the organs on ice in a tube containing HBSS solution.
SECTION: Tissue collection
9. Open the stomach by cutting on the lesser curvature and open the duodenum along the mesentery line. 
SECTION: Tissue collection
10. Wash the tissue in HBSS solution in a petri dish to clean and remove the food.
SECTION: Tissue collection
11. Place the tissue in a Petri dish coated with Sylgard and orient it mucosa up.
SECTION: Tissue collection
12. Grasp the right and left edges of the tissue and pin with 0.20 mm pins.
 

SECTION: Tissue collection
13. Fix the tissue in 4% PFA 7 min at 4 °C in the Petri dish coated with Sylgard.
SECTION: Tissue collection
14. Wash in PBS.
SECTION: Tissue collection
15. Unpin the tissue and keep it stored at 4 °C in 1X PBS containing 0.1% Sodium Azide (NaN3) before dissecting.
SECTION: Tissue collection
14.1. Wash in PBS for 5 min. (1/5)

SECTION: Tissue collection
14.2. Wash in PBS for 5 min. (2/5)
SECTION: Tissue collection
14.3. Wash in PBS for 5 min. (3/5)
SECTION: Tissue collection
14.4. Wash in PBS for 5 min. (4/5)
SECTION: Tissue collection
14.5. Wash in PBS for 5 min. (5/5)
SECTION: Microdissection of longitudinal muscle and myenteric plexus
16. Add PBS 1X to a Petri dish coated with Sylgard, place the tissue on it and orient tissue with the mucosal layer facing up. Grasp the right and left edges of the tissue and pin them.
 

SECTION: Microdissection of longitudinal muscle and myenteric plexus
17. Under a stereo microscope, scratch the mucosa with the back of curved forceps. 
SECTION: Microdissection of longitudinal muscle and myenteric plexus
18. Using forceps with fine tips, remove the mucosal and submucosal layer until you expose the circular muscle. 
SECTION: Microdissection of longitudinal muscle and myenteric plexus
19. Peel away the circular muscle with fine forceps to uncover the myenteric plexus.
SECTION: Microdissection of longitudinal muscle and myenteric plexus
20. With a micro-scissor, cut small segments of the dissected tissue containing the longitudinal muscle and the myenteric plexus (LMMP).
SECTION: Microdissection of longitudinal muscle and myenteric plexus
21. Store the LMMP preparation at 4 °C in 1X PBS containing 0.1% Sodium Azide (NaN3) until performing immunofluorescence.
SECTION: Immunofluorescence staining on whole mount tissues
22. In a 96-well plate, add 200 µL of blocking solution containing 0.1% PBS/NaN3, 10% FBS and 0.5% Triton X-100 per well needed for the number of tissues. Using fine forceps, transfer each tissue into a separate well with the blocking solution and incubate for 120 min at Room temperature on a shaker.
SECTION: Immunofluorescence staining on whole mount tissues
23. Dilute primary antibodies in the blocking solution.
SECTION: Immunofluorescence staining on whole mount tissues
24. Add 200 µL of primary antibodies solution in new empty wells, transfer each tissue in separate wells and incubate with the primary antibodies 120 min at 4 °C on a shaker.

SECTION: Immunofluorescence staining on whole mount tissues
25. Wash in PBS 1X (by adding 200 µL of PBS 1X in new wells and transferring each tissue separately).

SECTION: Immunofluorescence staining on whole mount tissues
26. Dilute secondary antibodies at 1:500 in the blocking buffer.
SECTION: Immunofluorescence staining on whole mount tissues
27. Add 200 µL of secondary antibodies solution in new empty wells, transfer each tissue in separate wells and incubate for 120 min at Room temperature on a shaker.

SECTION: Immunofluorescence staining on whole mount tissues
28. Wash in PBS 1X (by adding 200 µL of PBS 1X in new wells and transferring each tissue separately).
SECTION: Immunofluorescence staining on whole mount tissues
29. Place the tissue on the slide and mount it between slide and coverslip with prolong gold w/ DAPI.
SECTION: Immunofluorescence staining on whole mount tissues
30. Let it dry 120 min at Room temperature in a slide folder.
SECTION: Immunofluorescence staining on whole mount tissues
25.1. Wash in PBS for 5 min. (1/5)
SECTION: Immunofluorescence staining on whole mount tissues
25.4. Wash in PBS for 5 min. (4/5)
SECTION: Immunofluorescence staining on whole mount tissues
25.3. Wash in PBS for 5 min. (3/5)
SECTION: Immunofluorescence staining on whole mount tissues
25.2. Wash in PBS for 5 min. (2/5)
SECTION: Immunofluorescence staining on whole mount tissues
25.5. Wash in PBS for 5 min. (5/5)
SECTION: Immunofluorescence staining on whole mount tissues
28.1. Wash in PBS for 5 min. (1/5)
SECTION: Immunofluorescence staining on whole mount tissues
28.2. Wash in PBS for 5 min. (2/5)
SECTION: Immunofluorescence staining on whole mount tissues
28.3. Wash in PBS for 5 min. (3/5)
SECTION: Immunofluorescence staining on whole mount tissues
28.4. Wash in PBS for 5 min. (4/5)
SECTION: Immunofluorescence staining on whole mount tissues
28.5. Wash in PBS for 5 min. (5/5)