Introduction
- Amyloid refers to a group of abnormally folded protein aggregates that accumulate in tissues, leading to amyloidosis.
- Detecting amyloid deposits in tissues is crucial for diagnosing amyloidosis and understanding disease pathology.
- Special stains help differentiate amyloid from other extracellular deposits and confirm its presence microscopically.
Special Stains for Amyloid
Alkaline Congo red stain
- The method obviates the need for a differentiation step by including a high concentration of sodium chloride.
- This reduces background electrochemical staining whilst enhancing hydrogen bonding of Congo red to amyloid, resulting in a progressive and highly selective technique.
- The solutions should be discarded after 1 month.
- Ready-to-use commercial reagents and kits have up to 12 months of shelf life.
Fixation
Not critical
Stock solutions
1% aqueous sodium hydroxide
Stock solution a
Saturated sodium chloride in 80% ethanol.
Stock solution b
Saturated Congo red in 80% ethanol saturated with sodium chloride.
Leave to stand overnight before use.
Working solutions
A. To 100ml of stock solution add 1ml of 1% aqueous sodium hydroxide and filter.
B. To 100ml of stock solution b add 1ml of 1% aqueous sodium hydroxide and filter.
Prepare just before use.
Procedure
1. Take sections to water, removing pigment where necessary.
2. Stain nuclei in alcoholic alum hematoxylin (e.g. Mayer’s) and blue.
3. Immerse in alkaline sodium chloride solution for 20 minutes (working solution A).
4. Drain briefly, it is important NOT to blot.
5. Transfer directly to the alkaline Congo red solution for 20 minutes (working solution B).
6. Rinse briefly in alcohol, clear and mount.
Results
Amyloid – Red
Elastic and collagen fibers, eosinophil granules – Pink
Nuclei – Blue
Highman’s Congo red stain
This simple method has found wide application. The solutions are relatively stable and the method affords a high degree of selectivity in practiced hands.
Fixation
Not critical.
Solutions
0.5% Congo red in 50% alcohol
0.2% potassium hydroxide in 80% alcohol
Procedure
1. Take sections to water, removing pigment where necessary.
2. Stain in Congo red solution for 5 minutes.
3. Differentiate with the alcoholic potassium hydroxide solution for 3–10 seconds.
4. Wash in water, stain nuclei in alum hematoxylin, differentiate and blue.
5. Dehydrate, clear and mount.
Results
Amyloid, elastic fibers, eosinophil granules – Red
Nuclei – Blue
Congo red stain
This method has no differentiation step as Congo red is applied in an alkaline alcoholic solution. Carazzi’s hematoxylin was originally used as a counterstain, but any alum hematoxylin will suffice.
Fixation
Not critical.
Preparation of staining solution
Dissolve 0.5 g potassium hydroxide in 50 ml distilled water, add 200 ml absolute alcohol and add Congo red until saturated (about 3g). Stand overnight before use and discard after 3 months.
Procedure
1. Take sections to water, removing pigment where necessary.
2. Stain in filtered Congo red solution for 25 minutes.
3. Wash in distilled water, then running tap water for 5 minutes.
4. Counterstain nuclei in Carazzi’s hematoxylin for 1 minute.
5. Blue, differentiate the hematoxylin if necessary, blue.
6. Dehydrate, clear, and mount.
Results
Amyloid, elastic tissue, eosinophil granules – Red
Nuclei – Blue
Sirius red stain
- Llewellyn’s method modifies Sweat’s and uses the cotton dye Sirius red F3B – not to be confused with Sirius red 4B, which does not stain amyloid.
- Although this method is considerably simpler than the original technique, the staining solution does not keep well and tends to precipitate out of the solution.
Fixation
Not critical.
Preparation of solution
Dissolve 0.5 g Sirius red F3B in 45 ml distilled water. Add 50 ml absolute alcohol and 1 ml 1% sodium hydroxide. Stirring the solution vigorously, slowly add just sufficient 20% sodium chloride (more than 4 ml) to produce a fine precipitate when viewed against strong backlighting. Leave to stand overnight and filter.
Procedure
1. Take sections to water, removing pigment where necessary.
2. Stain nuclei in an alum hematoxylin, differentiate, and blue.
3. Rinse in water and then 70% ethanol.
4. Treat with Sirius red solution for 1 hour.
5. Wash in tap water for 10 minutes.
6. Dehydrate, clear and mount.
Results
Amyloid, elastic, eosinophil, and Paneth cell granules – Red
Nuclei – Blue