1.0 AMYLOID

1.1 ALKALINE CONGO RED FOR AMYLOID
(Puchster, Sweat & Levine, 1962)
Amyloidosis is a disease characterised by the deposition of amorphous eosinophilic, predominantly extracellular, material (amyloid) in various body tissues forming confluent masses and progressively replacing the parenchymatous cells of vital organs, resulting in gradual loss of function. Amyloid is composed of 95% protein + 5% carbohydrate.

PRINCIPLE
Congo Red dye forms non-polar hydrogen bonds with amyloid and red to green birefringence occurs when viewed by polarised light due to parallel alignment of the dye molecules on the linearly arranged amyloid fibrils. The Alkaline Congo Red Technique uses high concentrations of sodium chloride which act as ionic competitors for the dye thus eliminating background electrochemical (polar) staining while enhancing the hydrogen (non-polar) binding of Congo Red and amyloid.

REFERENCES
P 92 H.C. Cook, Histological Demonstration Techniques P 163 Bancroft JD & Stevens A Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Standard paraffin section Frozen section (gives greater intensity of staining)

CONTROL
Tissue positive for amyloid.
Note: 1. Unstained control sections tend to lose reactivity if stored for long periods Tissue containing massive deposits, gives less intense reaction than small newly formed deposits.

REAGENTS
Stock solutions
(A) Stock alcoholic salt solution Saturated sodium chloride in 80% alcohol (keeps well)
(B) 1% aqueous sodium hydroxide Dissolve 1g sodium hydroxide in 100 mL distilled water
(C) Stock Congo Red solution
Caution: Possible teratogen - Minimise contact - Wear gloves and mask - Wash thoroughly after handling Make a saturated solution of Congo Red C.I. 22120 in stock alcoholic salt solution (solution A). Approx 0.2g of dye required to saturate 100 mL. Keeps well. Store in fridge. Working solutions Alcoholic sodium chloride-hydroxide Solution Place in a coplin jar
Stock alcoholic salt solution
(A) 50 mL 1% aqueous Sodium hydroxide
(B) 0.5 mL Use within 20 minutes of preparation. Congo Red Working Solution Place in a coplin jar

Stock Congo Red solution
(C) 50 mL 1% aqueous sodium hydroxide (B) 0.5 mL Mix and filter, use within 20 minutes of preparation.
N.B. Takes approximately 20 minutes to filter.

PROCEDURE
1. Deparaffinise test and control sections and hydrate to water.
2. Stain nuclei with an alum haematoxylin (use linear stainer).
3. Wash in water.
4. Differentiate in 0.15% acid-alcohol approximately 10 seconds (in linear stainer).
5. Wash in water.
6. "Blue" in Lithium carbonate solution pH 7.8 to 8.6 (in linear stainer).
7. Wash in water.
8. Treat in working Alcoholic Sodium chloride-hydroxide solution for 20 minutes.
9. Drain.
10. Treat with working Congo red solution for 20 minutes.
11. Drain well.
12. Dehydrate rapidly in alcohols.
13. Clear and mount.

RESULTS

Amyloid (eosinophil granules, elastin) Dull red Nuclei Blue Background Clear Polarised light: Amyloid Green birefringence

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