Congo Red birefringence assay was developed for the examination of in vivo amyloid and was later applied in the examination of in vitrosamples. Many things other than amyloid exhibit birefringence; buffer salts (phosphate and urea) are inherently birefringent and appear as intense white birefringence, hair or other fibers exhibit birefringence but are often multicolored. This technique is relatively subjective and a known fibrillar sample should be used as a control.
Congo Red spectrophotometric assay is more objective, less prone to misinterpretation, and can be easily combined with the microscopic analysis.
Procedures for Congo Red birefringence assay:
1. Prepare the staining solution: prepare a solution of 80% EtOH: 20% DDI water and add a saturating amount of NaCl. Stir this solution for a few minutes and filter away the excess NaCl. Add a saturating amount of Congo Red, stir, and filter to obtain thefinal working solution. Congo Red can be used straight from the manufacturer or can be recrystallized from 50% EtOH: 50% DDI water prior to use. The staining solution should be used immediately or stored in 0.05% sodiumazide to inhibit bacterial growth.