 |
|
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.
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
Procedure:
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.
Results:
Amyloid, elastic fibers, eosinophil granules -------------
red
Nuclei
-------------------------------------------------------- blue
Positive Controls:
Know amyloidosis containing tissue (i.e. Alzheimer’s disease).