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Electron Microscopy Sciences

Technical Data Sheets

Genta Robason and Graham Stain for Simultanious 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:

Mucus cells Brownish yellow
Lamina propria ground substance Light yellowish or greyish
Smooth muscle fibers Light pink
Cytoplasm of neutrophils Pale pink
Eosinophils Intense red
Nucleus Dark brown
Intestinal metaplasma Bright blue

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.

Online Ordering

Genta. Robason and Graham Stain for Simultanious Visualization of Helicobacter Pylori and Gastric Morphology is available online from the EMS Catalog. For ordering or product information, click here.