Stomach tissue is collected from adult Sprague-Dawley male and female rats. Rats are supplied with food and water ad libitum prior to any tissue collection. All procedures were approved by The Florey Institute of Neuroscience and Mental Health Animal Ethics Committee.

Animals are deeply anesthetized with either an intraperitoneal injection of pentobarbital sodium (100mg/kg) or an intraperitoneal injection of a mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg) prior to being perfused transcardially with phosphate buffered saline (PBS: 0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.2) followed by fixative.

Different fixation methods are completed to allow for comparisons, these fixation methods are the following; 4% paraformaldehyde (Sigma Aldrich, USA), 10% neutral buffered formalin (Trajan, Melbourne, Australia) or Zamboni’s fixative (2 % formaldehyde plus 0.2 % picric acid in 0.1 M sodium phosphate buffer, pH 7.0). Some samples are placed into PBS containing nicardipine (1 µm) for 30-40 mins at room temperature before fixation.

Place tissue into histology cassettes in desired orientation and dehydrate through graded ethanol to histolene before embedding samples in paraffin.

Cut sections (5 μm) on microtome, dry sections at 37.7 ºC and stain with haematoxylin and eosin (H&E).

This is completed using a Leica Autostainer XL and Leica CV5030 coverslipper

Haematoxylin and Eosin staining steps are as follows:

1. Histolene - 5 minutes

2. Histolene - 1 minute

3. Absolute Alcohol - 1 minute

4. Absolute Alcohol - 1 minute

5. 70% Alcohol - 1 minute

6. Wash in running tap water

7. Stain in Mayer’s Haematoxylin - 6 minutes

8. Wash in running tap water - 1 minute

9. Differentiate in 0.3% Acid alcohol - 1-2 dips

10. Blue in Scott’s tap water (magnesium sulfate buffered with sodium bicarbonate) - 1 minute

11. Wash in running tap water - 3 minute

12. Check microscopically

13. Counter stain with Eosin - 3 minute

14. Rinse in running tap water - 1-2 dips

15. 70% Alcohol - 2 dips

16. Absolute Alcohol - 4 dips

17. Absolute Alcohol - 1 minute

18. Absolute Alcohol - 1 minute

19. Histolene - 1-2 minutes

20. Histolene - 1-2 minutes

21. Histolene - 1-2 minutes

22. Mount in DPX

Results:

Nuclei-blue

Cytoplasm, muscle, collagen, erythrocytes-pink/red

Masson’s trichrome staining is conducted manually by submerging slides into the solutions described in the steps below.

Masson's trichrome staining steps are as follows:

1. Bring sections to water.

2. Place sections in Bouin's fixative - 60 minutes at 60°C

3. Wash in running water - 10 minutes

4. Iron Haematoxylin

4.1 Weigert's A (I part) - 2 minutes

4.2 Weigert's B (I part) - 2 minutes

5. Wash in water.

6. 1% Ponceau 2 R- (2 parts) + 1 % Acid - (1 Part) - 5 minutes

7. Wash in water.

8. 1% Phosphomolybdic Acid - 3 minutes

9. Wash in water

10. 1%n - 5 minutes

11. Wash and leave in 1% Acetic Acid - 1 minute

12. 100% ethanol

13. Dehydrate, clear in xylene

14. Coverslip sections using DPX mounting media.

Results:

Nuclei-blue/black

Cytoplasm, muscle and RBC-red

Collagen-blue/green

Examine and image slides using an Axioplan microscope (Zeiss, Sydney, Australia).

A selection of fiducial points are chosen as sites to measure the thickness of the mucosal and muscle layers in rat stomach.

At each point three measurements of each muscle layer (mucosa, muscularis mucosae, longitudinal muscle, circular muscle, oblique muscle (where applicable)) are made, the average of these measurements is calculated.

Average these measurements for all equivalent fiducial points measured.