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

REAGENT

PURPOSE

MECHANISM OF STAINING

SOURCE OF ERROR

Harris’ Hematoxylin

Nuclear stain

 

Ionic bonding

Omitted: No nuclear stain.

Too short: Pale nuclear stain.

Too long: Nuclear details will be obscure.

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.

Too long: No effect.

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.

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