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Modified High pH Congo Red Staining Protocol for Amyloid
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.
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
Positive Controls:
Know amyloidosis containing tissue (i.e. Alzheimer’s disease).