Amyloid is an amorphous extracellular eosinophilic substance that can be present in various tissues and organs. The diseases caused by it are called amyloidosis. Amyloid is mainly composed of proteins, most of which are arranged in antiparallel β-pleated sheet structures. Under an electron microscope, amyloid appears as a fibrillary arrangement, presenting as a large number of unbranched extracellular filaments in pathological specimens, mostly arranged randomly. Histological methods for identifying amyloid include methyl violet staining, Congo red staining, and polarized light microscopy. Current research has found that the traditional methyl violet staining method has low sensitivity and poor specificity, while the classic and effective method is Congo red staining. In 1922, Bennhold discovered that Congo red could be used for the identification of amyloid in vivo and applied it to tissue sections.
Amyloid Staining Solution (Bennhold Congo Red Method) is mainly composed of Congo Red Staining Solution, Hematoxylin Staining Solution, etc. Its staining principle lies in that amyloid has a higher affinity for Congo red than other tissue structures, and its hydroxyl groups combine with the amino groups of Congo red, thus staining amyloid red. This staining method has stable performance and is a very classic method for amyloid staining. This reagent is for research use only and is not suitable for clinical diagnosis or other purposes.
A1508461
| Component | 5×50 mL | Storage | | A1508461A | Bennhold Hematoxylin Staining Solution
| 50 mL | RT. Store in the dark. | | A1508461B | Acid Ethanol Differentiating Solution
| 50 mL | RT | | A1508461C | Scott's Bluing Solution
| 50 mL | RT | | A1508461D | Congo Red Staining Solution
| 50 mL | RT. Store in the dark. | | A1508461E | Bennhold Differentiating Solution
| 50 mL | RT |
|
Materials to be Prepared by User
10% neutral formalin fixative, distilled water, graded ethanol, xylene or environment-friendly paraffin removal and clearing solution
Procedure (for reference only)
1. Routine fixation, usually with 10% neutral formalin fixative, followed by routine dehydration and embedding.
2. Cut sections with a thickness of 4 μm, and perform routine dewaxing to water using xylene or dewaxing and clearing solution.
3. Immerse in Bennhold Hematoxylin Staining Solution for 5 min.
4. Differentiate with Acid Ethanol Differentiating Solution for 2-5 s, immediately transfer to water to terminate differentiation, wash twice with water, and check under a microscope to ensure appropriate differentiation.
5. Rinse with tap water for 2 min.
6. Immerse in Scott's Bluing Solution for bluing, then rinse with tap water for 2 min.
7. Immerse in Congo Red Staining Solution for 30 min.
8. Rapidly differentiate with Bennhold Differentiating Solution for 15 s.
9. Transfer to freshly prepared 80% ethanol and differentiate until the red color is stable without fading, then rinse with tap water for 1-2 min.
10. Perform routine dehydration with graded ethanol, clear with xylene or dewaxing and clearing solution, and mount with neutral balsam.
Staining Results
Amyloid: Red
Cell Nucleus: Blue
Note: Under a polarized light microscope, amyloid exhibits yellowish-green birefringence.
Precautions
1. Ensure thorough dewaxing of sections; otherwise, the staining effect will be affected.
2. Store Acid Ethanol Differentiating Solution in a sealed container and use it as soon as possible after opening.
3. Prefer immersion staining when using Congo Red Staining Solution; if drop staining is adopted, place the sections in a moist chamber to prevent solution evaporation.
4. The differentiation step with Bennhold Differentiating Solution is critical. Always monitor the differentiation degree until no excess dye drips off. Short differentiation time will result in collagen fibers being stained red; over-differentiation will cause amyloid to decolorize. If over-decolorization occurs, rinse the sections and re-stain with Congo Red Staining Solution.
5. For your safety and health, wear a lab coat and disposable gloves during operation.
6. Use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.
ZJ26F0332657