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Congo Red Stain in Amyloidosis

Congo red stain in amyloidosis post we have briefly explained about Congo red stain method’s principle, requirements, Congo red staining for amyloid procedure, result with Label.

Congo Red Stain in Amyloidosis

Congo red staining for amyloid is for determining the presence of amyloid in tissue sections. There is a regular process as well as a microwave procedure. Reagents for Amyloid Stain are intended for “In Vitro Diagnostic Use.”

Amyloidosis is caused by an aberrant protein that accumulates between parenchymatous cells of connective tissue as a result of an immunologic disruption. Congo red, a metachromatic anionic dye, can be used to identify amyloid in tissue.

Principle

The Congo red Amyloid Stain is used to determine whether or not tissue slices contain amyloid. Amyloid Stain Reagents are designed for “In Vitro Diagnostic Use.”

Amyloidosis is caused by an abnormal protein that builds up between connective tissues parenchymatous cells as a result of an immune system malfunction. A metachromatic anionic dye called Congo red can be used to identify amyloid in tissue.

Congo red staining for amyloid principle is based on hydrogen bridge bonds forming with the substrate’s carbohydrate component. Congo red is an anionic dye that can deposit itself on amyloid fibrils, causing them to show a distinct dichroism when exposed to polarised light.

Under transmitted light, the Congo red-stained tissue appears orange-red; however, under polarised light, the amyloid deposits emerge as vivid green double-refraction pictures against a dark backdrop.

Under polarised light, other structures stained by Congo red, such as collagen, are not visible. When the paraffin sections utilised are too thin (5m) or the tissue is too highly coloured, Congo red staining for amyloid can be challenging.

Requirements

Congo Red Stain

1. Congo Red Stock Solution: Congo red – 0.3 g, Sodium chloride – 0.3 g, 80% Alcohol – 100 ml.

2. 1% Sodium Hydroxide: Sodium hydroxide – 1 g, Distilled water – 100 ml.

3. Congo Red Working Solution: 1% Sodium hydroxide – 0.5 ml, Congo red stock solution – 50 ml, Mix well, filter and use within 20 minutes.

4. Alkaline Alcohol Solution: 1% Sodium hydroxide – 1 ml, 50% alcohol – 100 ml

Storage and Stability

Reagents for Amyloid Stain should be kept at room temperature (18–26°C). Congo Red Solution and Sodium Chloride Solution, Alcoholic are stable until the expiration date indicated on the label.

Titration can be used to check the molarity of Sodium Hydroxide Solution. Precipitate in Sodium Hydroxide Solution, Alcoholic Solution, or Congo Red Solution has no effect on its application.

Procedure

1. Deparaffinize and hydrate sections to distilled water.

2. Stain in congo red working solution for 10 minutes.

3. Rinse in distilled water. 

4. Differentiate quickly (5-10 dips) in alkaline alcohol solution.

5. Rinse in tap water.

6. Counterstain in Gill’s hematoxylin for 30 seconds.

7. Rinse in tap water for 2 minutes.

8. Dip in ammonia water for 30 seconds or until sections turn blue.

9. Rinse in tap water for 5 minutes.

10. Dehydrate through 95% alcohol, 100% alcohol

11. Clear in xylene and mount with resinous mounting medium. 

Result

Congo red Stain

Congo red staining for amyloid: Amyloid, elastic tissue, eosinophil granules: Red and Nuclei: Blue  

Limitations

1. Congo red staining for amyloid might be challenging, especially if the paraffin sections used are very thin (5 m is recommended for Congo red staining for amyloid).

2. In the event of an overabundance of ethanol incubation during the dehydration process, the Congo red dye may be washed out of the specimen tissue, resulting in specimen decolorization.

3. As a precaution, congo red staining for amyloid protocol’s dehydration durations should be closely followed to avoid false-negative findings.

Further Readings

Reference