Congo Red (Direct Red 28) is an azo dye. It binding been used as a diagnostic test for the presence of amyloid in tissue sections.

Congo Red histochemical stain may serve as a tool to investigate if aggregates in mutant cells have misfolded β-pleated sheet secondary structures [1]. Congo Red dye specifically binds to crossed β-pleated sheet structures. While wild-type HSPB1 maintains protein homeostasis by binding proteins in non-native conformations and preventing aggregation, the T139M mutant fails in this function, resulting in the accumulation of misfolded proteins targeted by Congo Red for intercalation between the β-pleated sheet structures.

Instructions
I. Solution preparation
1. Congo Red working solution: Dissolve Congo Red in distilled water or an appropriate buffer, usually at a concentration of 0.5–1% (w/v).
When staining for amyloid, it is recommended to add a high concentration of sodium chloride (such as 80% saturated sodium chloride solution) to enhance the selectivity of the dye for amyloid.
2. Alkaline alcohol differentiation solution (for differentiation after staining): Dissolve 1% NaOH in 50% ethanol and set aside.
3. Contrast staining solution (such as Mayer's hematoxylin): Used to stain cell nuclei and enhance contrast.
II. Congo Red operation steps in amyloid staining
1. Tissue section preparation: Fix tissue samples with a fixative (such as 10% neutral buffered formaldehyde) and make paraffin sections (4–6 µm thick). Dewax the sections and hydrate the sections into water through a gradient of ethanol (such as 100%, 95%, 70%).
2. Staining step: Immerse the slices in Congo Red working solution, usually for 20–30 minutes. Then rinse the slices gently with running water to remove unbound dye.
3. Differentiation step: Immerse the stained slices in alkaline alcohol differentiation solution (about 30 seconds to 1 minute) to reduce nonspecific background staining. Rinse the slices with running water and observe the staining effect.
4. Contrast staining: Mayer's hematoxylin can be used to stain the slices (about 1–3 minutes), then rinse with running water and return to blue with 0.5% ammonia water.
5. Dehydration and sealing: Use gradient ethanol (such as 70%, 95%, 100%) for dehydration in sequence, then use xylene to transparent the slices and seal the slices with neutral gum.
6. Microscope observation: Amyloid protein appears orange-red after Congo Red staining, and typical green birefringence can be seen when observed under a polarizing microscope, which is the key feature of Congo Red's specific detection of amyloid protein.
Notes
1. Fresh dye: Congo Red working solution needs to be replaced regularly to ensure the staining effect and avoid the dye from becoming ineffective due to long-term use.
2. Background staining: The differentiation step is crucial to reduce nonspecific background staining, and the differentiation time or alkaline alcohol concentration can be optimized.
3. Environmental control: Staining should be performed at room temperature and avoid long-term exposure to high temperature.
4. Photosensitivity: Congo Red dye and stained sections should be kept away from light to avoid photodegradation.