Description: This high pH 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:
Congo Red Stock Solution:
Congo red (Sigma, Cat# C-6277) --------- 0.3 g
Sodium chloride ---------------------------- 0.3 g
      80% Alcohol --------------------------------- 100 ml
1% Sodium Hydroxide:
Sodium hydroxide ------------------------- 1 g
Distilled water ----------------------------- 100 ml
Congo Red Working Solution:
1% Sodium hydroxide ---------------------- 0.5 ml
Congo red stock solution ------------------ 50 ml
Mix well, filter and use within 20 minutes.
Alkaline Alcohol Solution:
1% Sodium hydroxide --------------------- 1 ml
50% alcohol -------------------------------- 100 ml
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 (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.
Results:
Amyloid, elastic tissue, eosinophil granules ------- red
      Nuclei -------------------------------------------------- blue Â
Positive Controls:
Know amyloidosis containing tissue (i.e. Alzheimer’s disease).