Modified from Hopkins Neuropathology 8/09

CONGO RED STAIN

Fibrillar amyloid plaques will stain pale red with CR, nuclei will stain pale lavender/blue with hematoxylin, background should be clear. Plaques stained with CR should appear red/green birefringent under polarized light.

Hematoxylin Solution

Add each chemical one at a time, stirring thoroughly after each. Make sure each ingredient is fully dissolved before adding the next. Stir for an additional 2 hours once all components are added, then allow the stain to ripen in sunlight for at least one month prior to use. The final color of the stain should be reddish-violet. Store at room temp., shelf life is approx 6 mo.

0.5 g Hematoxylin (Sigma #H9627)

500 ml H2O

0.1 g Sodium Iodate (Sigma #S4007)

25 g Ammonium Aluminum Sulfate (Malinckrodt #3212)

0.5 g Citric acid (Malinckrodt #0627)

25 g Chloral hydrate (Sigma #C8383)

NOTE: Class IV restricted substance, requires DEA license to purchase

2 drops Glacial acetic acid (final pH should be approx. 2.5)

NOTE: When used as a counterstain, this stain should be diluted 5-10-fold in acid alum (the base without the dye)

Stock Congo Red Solution (0.25% Congo Red in 80% EtOH)

Solution may require several days of stirring to dissolve (one day minimum); even with protracted stirring undissolved particulates will remain. This solution is stable for six months.

100 ml H2O

400 ml Ethanol, 200 proof

15 g NaCl

1.25 g Congo Red dye (Sigma #C6277, histology grade)

Working Congo Red Solution

Filter solution twice through glass wool before use. Do not substitute filter paper – only glass wool retains the dye’s birefringence. Use working solution immediately and discard after use.

100 ml Stock Congo Red solution

1.0 ml 1% NaOH

Procedure

1. Filter hematoxylin solution using fluted Whatman paper.

2. Rehydrate sections in running lukewarm tap water for 20-30 minutes.

3. Stain in 10-fold diluted hemotoxylin for 15-20 minutes.

4. Rinse in running lukewarm tap water for 5-15 minutes.

5. Stain in congo red solution for 50-60 minutes.

6. Rinse in running lukewarm tap water for 15-20 minutes.

7. Dehydrate through an ascending alcohol series (70%, 95%, 100%) into xylene.

8. Coverslip with Permount.