Jone's Methenamine Silver
Classification: connective tissue stain
Purpose: stain glomerular basement membrane
Staining principle: argentaffin metallic impregnation
Positive controls: aorta, skin, lung, internal control
Well Stained Slide
Results:
Glomerular basement stains brown-black
REAGENT |
PURPOSE |
MECHANISM OF STAINING |
SOURCE OF ERROR |
Periodic acid |
Mild oxidizer which does not oxidize
anything but the basement membrane |
Exposes reactive aldehyde groups |
Omitted: Basement membrane will not be
demonstrated. |
Too short: Basement membrane may have patchy
staining. |
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Too long: Does not affect the staining. |
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Methenamine silver |
Impregnation (Silver nitrate,hexamine and borax) |
Silver binds with the aldehyde groups (reactive groups) Aldehyde groups will reduce silver |
Omitted: Basement membrane will not be
demonstrated. |
Too short: Patchy staining of basement
membrane may be seen. |
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Too long: Difficulty in seeing the details
in glomerulus. |
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Gold chloride |
Toning (optional) |
Substitute silver with gold by ion exchange Removes any brown color in other tissue components |
Omitted: Collagen will remain golden brown. |
Too long: Production of red tones due to overtoning. Will
start to remove silver staining. |
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Too short: No effects on the staining. |
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Sodium thiosulphate (hypo) |
Fixation |
Removes unreacted silver |
Omitted: Causes non-specific blackening of
tissue over time. |
Too long: No effects on the staining. |
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Too short: Causes blackening of tissue over
time. |
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Light green |
Counterstain |
|
Omitted: No other tissue components demonstrated. |
Too long: Basement membrane obscured. |
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Too short: Other tissues stain lightly and
no detail seen. |
Poor Stain
Counterstain is too dark.
Basement membrane is obscured.
Well Stained Slide
Details of basement membrane of kidney glomerulus are visible.
Well Stained Slide
Details of basement membrane of kidney glomerulus are visible.
Special Considerations
When methemine silver complex is used the aldehydes reduce the silver of the methemine silver and form a metallic silver. Therefore, no extranous reducer is required.
Timing is important when working with silver at 70 degrees celsius, the solution is good for 60 to 75 minutes and this should be done without preheating.
Toning with gold chloride is an optional step.
Renal tubular basement membrane will be stained black before basement membranes. We are looking for glomerular basement membrane which should be appear brown-black.
No metal forceps should be used when working with this stain.
This stain can not be removed from tissue after fixation step. Before the fixation we can remove the stain with iron alum.
Other Sources of Error
- black precipitates on the slides because of silver solution.
- formation of precipitates due to checking the slides too often.
- uneven staining due to cooling the slides too much
- sharper staining and less background staining may be achieved with microwave usage.
Reference
Officer B. HIML251 Lecture notes: Silver Stains. Jone's Methenamine Silver, March 26, 2009