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MODIFIED PUCHTLER CONGO RED AMYLOID METHOD
WITH/WITHOUT POTASSIUM PERMANGANATE OXIDATION
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Amyloid material deposition in small vessels viewed in two different phase of light show reciprocal color patter ("apple green" and orange) (Courtesy of Dr. Zhenhong Qu) |
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FIXATION: 10% buffered neutral formalin.
TECHNIQUE: Paraffin sections cut at 6 μm.
SOLUTIONS:
Acidified Potassium Permanganate Solution
Potassium permanganate -------------------------------------------------------- 0.3 gm
Distilled water ------------------------------------------------------------------- 100.0 ml
Sulfuric acid --------------------------------------------------------------------- 0.2 ml
1.0% Oxalic Acid
Oxalic acid ----------------------------------------------------------------------- 1.0 gm
Distilled water ------------------------------------------------------------------- 100.0 ml
Modified Weigert’s Iron Hematoxylin
Solution A
Hematoxylin, C.I. 75290 ------------------------------------------------------- 2.0 gm
Alcohol, 90% -------------------------------------------------------------------- 100.0 ml
Solution B
Ferric chloride, FeCl3 6H2O, 62% aqueous ------------------------------------ 4.0 ml
Distilled water ----------------------------------------------------------------- 95.0 ml
Hydrochloric acid, concentrated ---------------------------------------------- 1.0 ml
For use mix equal parts of Solution A and Solution B.
0.5% Acid Alcohol Solution
Alcohol, 70% ----------------------------------------------------------------- 1000.0 ml
Hydrochloric acid, concentrated ---------------------------------------------- 5.0 ml
Sodium Chloride-Alcohol Solution
Sodium chloride ----------------------------------------------------------------- 2.5 gm
Distilled water ------------------------------------------------------------------- 50.0 ml
Absolute alcohol ---------------------------------------------------------------- 50.0 ml
Dissolve the sodium chloride in the distilled water and then add
the absolute alcohol. This solution is stable for several months.
1% Potassium Hydroxide Solution
Potassium hydroxide ------------------------------------------------------------ 1.0 gm
Distilled water ------------------------------------------------------------------ 100.0 ml
Congo Red Solution
Sodium chloride – alcohol solution ---------------------------------------------- 50.0 ml
Congo red, C.I. 22120 ---------------------------------------------------------- 0.1 gm
1% Potassium hydroxide ------------------------------------------------------- 0.5 ml
Dissolve the Congo red in the sodium chloride-alcohol solution, then add the potassium hydroxide. Filter through Whatman #4 filter paper. Prepare just before use. This solution is stable for about one month.
In unoxidized sections, amyloid stains pink to red and demonstrates apple-green birefringence with polarizing microscopy and fluoresces orange to red when viewed with fluorescence microscopy. In oxidized sections, secondary (AA) amyloid shows diminished staining and fluorescence and does not exhibit green birefringence. Primary (AL) amyloid will retain the stain.
Amyloidosis is a group of diseases involving the deposition of insoluble polymerized protein filaments (amyloid) in the interstitial spaces of blood vessels and in various organs including heart, kidneys, lung and liver. Two major types of amyloid exist: primary (AL) and secondary (AA) amyloid. Primary amyloid is characterized by deposition of enzymatically altered immunoglobulin kappa or lambda light chains. Secondary amyloid comprises an enzymatically altered serum protein, which is made in the liver. Deposition of AA amyloid is usually seen in response to chronic inflammatory processes including pulmonary infection, tuberculosis, rheumatoid arthritis, osteomyelitis, leprosy and an occasional neoplasia.
Amyloid can be stained with several dyes including thioflavin T, thioflavin S, crystal violet, methyl violet, sirius red, and the dye of choice, Congo red. Congo red stains amyloid-oriented filaments by deposition of the linear dye molecules with their long axis parallel to protein filaments. This subsequently shows an apple-green birefringence when viewed by polarizing microscopy. We have found that the optimal way of viewing Congo red stained amyloid is by fluorescence microscopy. Small or weakly stained deposits of amyloid, which are often difficult to see by light or polarization microscopy, are readily apparent by fluorescence microscopy. We have, furthermore, not seen significant orange-red fluorescence from any tissue components other than amyloid, except for elastic fibers and coarse collagen fibers.
In this method a modified Weigert’s iron hematoxylin is used as the counterstain instead of Harris’ alum hematoxylin as prescribed by Puchtler. Differentiation of the iron hematoxylin with acid alcohol results in a clear background that allows better visualization of amyloid by light microscopy. The iron hematoxylin also quenches background fluorescence, thus facilitating viewing with a fluorescence microscope.
Churukian, C.J.: Improved Puchtler's Congo Red Method for Demonstrating Amyloid. J. Histotechnol. 23:139-141, 2000.
Churukian, C.J. and Schenk, E.A.: Eastwood Congo red method for demonstrating amyloid. J. Histotechnol. 11:105-107, 1988.
Wright, J.R., Calkins, E. and Humphrey, R.L.: Potassium permanganate reaction in amyloidosis. Lab. Invest. 36:274-281, 1977.
Puchtler, H., et al.: On the binding of Congo red by amyloid. J. Histochem. Cytochem. 10:355-363, 1962.
Image Examples:
Diagnostic Application: