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Bennhold’s Congo Red Staining Protocol for Amyloid
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Bennhold’s Congo Red Staining Protocol for Amyloid

Description: This Bennhold's Congo Red stain is used for the detection of amyloid on formalin-fixed, paraffin-embedded tissue sections with amyloidosis, and may be used for frozen sections as well. The amyloid deposits will be stained red and the nuclei will be stained blue. The thickness of sections is usually 5 um. But in case of inadequate amyloid deposits, 10um thick sections will be more satisfactory. Congo red stains amyloid in tissue sections.

Fixation: 10% formalin.

Section: paraffin sections at 5 um (in case inadequate amyloid deposits, use 10 um thick sections).

Solutions and Reagents:  

1% Congo Red Solution:

      Congo red (Sigma, Cat# C-6277) ------ 1 g

      Distilled water --------------------------- 100 ml

1% Sodium Hydroxide:

      Sodium hydroxide ------------------------ 1 g

      Distilled water ---------------------------- 100 ml

Alkaline Alcohol Solution:

      1% Sodium hydroxide  -------------------- 1 ml

      50% alcohol -------------------------------- 100 ml


1.   Deparaffinize and hydrate sections to distilled water.

2.   Stain in Congo red solution for 30-60 minutes.

3.   Rinse in distilled water.  

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

5.   Rinse in running tap water for 5 minutes.

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

7.   Rinse in tap water for 1 minute.

8.   Dip in ammonia water (add a few drops of ammonium hydroxide to tap water and mix well) 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.


      Amyloid, elastic fibers, eosinophil granules ------------- red

      Nuclei -------------------------------------------------------- blue  

Positive Controls:

      Know amyloidosis containing tissue (i.e. Alzheimer’s disease).

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