2. Air-dry 10µL of the aggregated protein solution onto a glass microscope slide.
3. Place 200–400µL of the staining solution onto dried protein sample. Wait for seconds and then blot away the excess solution with a lint-free Kimwipe(or hardened filter paper), being careful not to touch the sample, and allow the stained sample to dry at room temperature.
4. Examine the stained sample using polarized light microscopy.The microscope should have high quality,strain-free lenses for optimal performance. If the polarizersare aligned, the material stained with Congo Red will appear reddish pink (the affinity for CongoRed is known as congophilia). If the polarizers are crossed at a 90 angle to each other, the background of the sample will turn black. Any bright spots that appear are a result of birefringence (the sample bends the light in such a way that it can pass through the upper polarizer to reach your eye).The detection of yellow/green birefringence is considered a positive result for the presence of amyloid.The absence of such birefringence is a negativeresult. The birefringence under crossed polarizers should match the areas of Congo Red staining observed under visible light.