FIXATION:        10% buffered neutral formalin

TECHNIQUE:        Paraffin sections cut at 6 microns.

SOLUTIONS:

Modified Weigert’s Iron Hematoxylin

(see Puchtler's Congo Red Amyloid Method)

0.5% Acid Alcohol Solution

(see Puchtler's Congo Red Amyloid Method)

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

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 hydroxide.  Filter through Whatman #4 filter paper.  This

       solution is stable for about one month.

1. Deparaffinize and hydrate slides to distilled water.
2. Place in modified Weigert’s iron hematoxylin for 10 seconds.
3. Wash briefly in running tap water and rinse in two changes of distilled water.
4. Place in acid alcohol solution for 5 seconds.
5. Wash in running tap water for 1 minute.
6. Rinse in two changes of distilled water.
7. Place in 95% alcohol for 5 seconds.
8. Place in Congo red solution for 20 minutes.
9. Dehydrate in three changes of 95% alcohol and four changes of absolute alcohol.
10. Clear in xylene, three or four changes.
11. Mount with synthetic resin.

Amyloid -------------------------------------------------------------        pink or red

Nuclei ---------------------------------------------------------------        black

Erythrocytes --------------------------------------------------------        pale orange

Eosinophil granules --------------------------------------------------                reddish orange

In Puchtler’s Congo red method, alum hematoxylin is used as the counterstain.  This tends to produce a faint, undesirable, pink background stain.  By using the modified Weigert’s iron hematoxylin, in place of alum hematoxylin, no background staining occurs.

Puchtler’s Congo red solution is prepared with a saturated solution of sodium chloride in 80% alcohol.  The sodium chloride suppresses non-specific staining of collagen, elastic and muscle thus enhancing the selectivity of the stain for amyloid.  In developing this modification of Puchtler’s method it was found that a less than saturated solution of sodium chloride in alcohol worked equally well as a saturated one in suppressing non-specific staining.  Congo red is more soluble in 50% alcohol than in 80% alcohol.  The more concentrated solution produces better staining of amyloid and has much greater stability than Puchtler’s Congo red solution.

In Puchtler’s method the slides are treated with saturated sodium chloride in 80% alcohol for 20 minutes prior to staining with Congo red.  We found this is unnecessary because it has no effect upon the final staining results.

Churukian, C.J.: Improved Puchtler's Congo Red Method for Demonstrating Amyloid. J. Histotechnol. 23:139-141, 2000.

Puchtler, H. et al.:  On the binding of Congo red amyloid.  J. Histochem. Cytochem. 10:355-363, 1962.

Eastwood, H. and Cole, K.R.:  Staining of amyloid by buffered Congo red in 50% ethanol.  Stain Technol. 46:208-209, 1971.

Brown, G.:  An Introduction to Histotechnology, New York, Appleton-Century-Crofts, 1978, pp. 318-319.