Congo Red
Classification: carbohydrate stain
Purpose: stain amyloid
Staining principle: hydrogen bonding
Mechanism of staining: non-ionic bonding
Positive controls: any tissue containing amyloid (kidney, prostate)
Well Stained Slide
Amyloid - deep red to pink
Elastic fibers - pale pink
Nuclei - blue
REAGENT |
PURPOSE |
MECHANISM OF STAINING |
SOURCE OF ERROR |
Harris’ Hematoxylin |
Nuclear stain |
Ionic bonding |
Omitted: No nuclear stain. |
Too short: Pale nuclear stain. |
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Too long: Nuclear details will be obscure. |
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Solution A |
Alkaline pretreatment Alkaline solution: NaOH NaCl 80% alcohol |
High pH increases the stain specificity for amyloid and
reduces background staining. |
Omitted: Background staining. |
Too short: Background staining. |
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Too long: No effect. |
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Solution B |
Amyloid stain: NaOH NaCl 80% alcohol Congo red (progressive) |
Selectivity of staining is due to composition of amyloid and
the contents of the staining solution |
Omitted: No demonstration of amyloid. |
Too long: No effect. |
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Too short: Pale staining of amyloid. |
Well Stained Slide
Amyloid - deep red to pink
Elastic fibers - pale pink
Nuclei - blue
Kidney
Polarizing Light Microscopy
Amyloid is capable of changing the direction of light through the filters of a polarizing microscope. The polarized light vibrates in only one plane and results in birefringence.
Amyloid - apple green birefringence
Amyloid may display a weak autofluorescence using non-polarizing light microscopy.
When confirming the presence of amyloid use a positive tissue control that has been freshly cut.
Special Considerations
Other stains can stain amyloid. Alcian Blue stains positive (blue) at pH 2.5 because of sialic acid glycoproteins present in amyloid. Hematoxylin and eosin stains amyloid as amorphous, eosinophilic fibers. Periodic Acid Schiff stains weakly positive (magenta). Masson Trichrome will stain amyloid light green, like collagen.
Filter and use Congo Red within 20 minutes of preparation.
Do not induce any water after the alkaline pretreatment is added.
References
Officer B. HIML251 Lecture notes: Congo Red, Toluidine Blue, and Oil Red O, March 11, 2009