Genta Robason and Graham Stain for Simultaneous Visualization of Helicobacter Pylori and Gastric Morphology

EMS Catalog #:26116-series

Fixation:

Formalin fixed

Sections:

Paraffin @ 4 um sections

Staining:

1. Deparaffinize and hydrate sections to distilled water.
2. Sensitize sections by placing them in Uranium Nitrate. 1%, Aqueous (#26116-01) at room temperature for 3 minutes and transfer them to distilled water.
3. Rinse slides in distilled water until the possibility of cross contamination is eliminated.
4. Place sections in Silver Nitrate, 1% (#26116-02) at room temperature and then heat then in a microwave oven to just below the boiling point.   DO NOT BOIL!
5. Rinse slides in 3 changes of distilled water.
6. Rinse in 2 changes of 95% alcohol.
7. Rinse in 2 changes of 100% alcohol.
8. Place slides in 2.5% Gum Mastic (#26116-03) for 5 minutes.
9. Air dry sections for 1 minute
10. Rinse in 2 changes of distilled water.
11. Reduce in reducing solution in a 450°C water bath for 10 to 15 minutes or until sections have developed satisfactorily with black or dark brown H-Pylori and a light yellow background. Check periodically under microscope.
    
    To prepare reducing solution
    - Gum Mastic, 2.5 % (#26116-03) - 10 ml
    - Hydroquinone, 2% (#26116-04) - 25 ml
    - Absolute Alcohol (#15055) - 5 ml
    - Silver Nitrate, 0.04% (#26116-02) - 2.5 ml
    Mix together - DO NOT FILTER!!
12. Rinse sections in water to stop reduction.
13. Stain in Alcian Blue, pH 2.5 (#26116-06) for 10 minutes.
14. Rinse in running tap water.
15. Stain sections in Harris' Hematoxylin (#26116-07) for 5 minutes.
16. Rinse in running tap water.
17. Quickly dip in Acid Alcohol (#26116-08).
18. Rinse in running tap water.
19. Rinse in Ammonia Water, 0.25% (#26116-09) until blue.
20. Rinse in running tap water.                            
21. Stain sections in Eosin Y, 1% (#26116-10) for 5 minutes.
22. Quickly rinse in tap water.
23. Rinse twice in 95% alcohol.
24. Rinse three times in absolute alcohol.
25. Rinse three times in Xylene (#23400) and then coverslip.

Staining Results:

H-pylori are easily recognized in the mucus, on the cell surface, and within the gastric pits.

References:

Genta PM., Robason GO, Graham DY- Simultaneous Visualization of Helicobactor Pylon and Gastric Morphology A New Stain Hum Pathol 25: 22 1-226, 1994.

Product Information

Genta. Robason and Graham Stain for Simultanious Visualization of Helicobacter Pylori and Gastric Morphology