TITLE: Histological quantification of thickness of the mucosal and muscle layers of the rat stomach
AUTHORS: Madeleine R. Di Natale, Lauren Patten, Martin Stebbing, John Furness

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The thickness, organization and relationships between the muscle layers of the rat stomach assist in understanding their function. Here we describe protocols for identifying, measuring and quantifying the muscle layers of the rat stomach using histological and microscopy techniques. </div><br/><div class = "text-block">We would like to acknowledge Phenomics Australia Histopathology and Slide Scanning Service, University of Melbourne. </div></div>

[STEPS]
?. [Tissue Collection]
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.
?. [Histological Staining]
Place tissue into histology cassettes in desired orientation and dehydrate through graded ethanol to histolene before embedding samples in paraffin.
?. [Histological Quantification ]
A selection of fiducial points are chosen as sites to measure the thickness of the mucosal and muscle layers in rat stomach.
?. [Histological Staining]
Haematoxylin and Eosin staining steps are as follows: 1. Histolene - 5 minutes2. Histolene - 1 minute 3. Absolute Alcohol - 1 minute 4. Absolute Alcohol - 1 minute 5. 70% Alcohol - 1 minute 6. Wash in running tap water7. Stain in Mayer’s Haematoxylin - 6 minutes 8. Wash in running tap water - 1 minute9. 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 minute12. Check microscopically13. Counter stain with Eosin - 3 minute 14. Rinse in running tap water - 1-2 dips15. 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
?. [Histological Staining]
Masson's trichrome staining steps are as follows: 1. Bring sections to water.2. Place sections in Bouin's fixative - 60 minutes at 60°C3. Wash in running water - 10 minutes4. Iron Haematoxylin4.1 Weigert's A (I part) - 2 minutes4.2 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 water10. 1%n - 5 minutes11. 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/blackCytoplasm, muscle and RBC-redCollagen-blue/green
?. [Tissue Collection]
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.
?. [Tissue Collection]
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.
Be careful with fixatives. Refer to the each SDS/MSDS and follow local safety guidelines set by your institute and manufacturers.
?. [Histological Staining]
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
?. [Histological Staining]
Masson’s trichrome staining is conducted manually by submerging slides into the solutions described in the steps below.
?. [Histological Quantification ]
Examine and image slides using an Axioplan microscope (Zeiss, Sydney, Australia).
?. [Histological Quantification ]
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.
?. [Histological Quantification ]
Average these measurements for all equivalent fiducial points measured.