Amyloid is an amorphous extracellular eosinophilic substance that can be present in various tissues and organs, and the diseases caused by it are referred to as amyloidosis. Amyloid is mainly composed of proteins, most of which are arranged in an antiparallel β-pleated sheet structure. Under an electron microscope, amyloid exhibits a fibrillary arrangement, appearing 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 demonstrated that the traditional methyl violet staining method has low sensitivity and poor specificity, while Congo red staining is a classic and effective approach. In 1922, Bennhold discovered that Congo red could be used for the identification of amyloid in vivo and applied this technique to tissue sections.
Amyloid Staining Solution (Puchtler Alkaline Congo Red Method) is mainly composed of Congo Red Staining Solution and Hematoxylin Staining Solution. Its staining principle lies in that amyloid has a higher affinity for Congo red than other tissue structures, and its hydroxyl groups bind to the amino groups of Congo red, thereby staining amyloid red. This staining method has stable performance and is a very classic technique for amyloid staining. This reagent is for research use only and is not suitable for clinical diagnosis or any other purposes.
A1508466
| Component | 4×50 mL | Storage |
| A1508466A | Hematoxylin Staining Solution
| 50 mL | RT. Store in the dark.
|
| A1508466B | Acid Ethanol Differentiating Solution
| 50 mL | RT |
| A1508466C | Sodium Chloride Solution
| 50 mL | RT |
| A1508466D | Congo Red Staining Solution
| 50 mL | RT. Store in the dark.
|
| A1508466E | Puchtler Alkalizing Solution
| 1 mL | RT |
Materials to be Prepared by User
1. 10% neutral formalin, distilled water, graded ethanol
2. Xylene or environment-friendly paraffin removal and clearing solution, neutral balsam, Scott's Bluing Solution
Procedure (for reference only)
1. Perform routine fixation. There is no special requirement for the fixative; 10% neutral formalin fixative is commonly used, 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 the sections in Hematoxylin Staining Solution for 5 min.
4. Differentiate with Acid Ethanol Differentiating Solution for 2-5 s, immediately transfer the sections 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 the sections in Scott's Bluing Solution or rinse with water for bluing, then rinse with tap water for 2 min.
7. Prepare alkaline sodium chloride solution at a ratio of sodium chloride solution: Puchtler alkalizing solution=100:1. Prepare it immediately before use. Immerse the sections in the alkaline sodium chloride solution for 20 min.
8. Prepare alkaline Congo red staining solution at a ratio of Congo red staining solution: Puchtler alkalizing solution=100:1. Prepare it immediately before use. Directly immerse the sections in the alkaline Congo red staining solution for 20 min.
9. Rinse gently with anhydrous ethanol, clear the sections with xylene or dewaxing and clearing solution, and mount them with neutral balsam.
Staining Results
Amyloid: Red
Cell Nucleus: Blue
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. Prepare alkaline sodium chloride solution and alkaline Congo red staining solution immediately before use, and try to use them within 20 min.
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 the results of subsequent experiments.
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