Amyloid is an amorphous extracellular eosinophilic substance that can be present in various tissues and organs, and the diseases caused by it are called amyloidosis. Amyloid is mainly composed of proteins, most of which are arranged in an antiparallel β-pleated sheet structure. Under an electron microscope, amyloid presents 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 shown 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. Later, the method was modified by Highman, resulting in improved staining efficacy.
The modified Highman Congo red staining, also known as methanol Congo red staining, mainly consists of Congo red staining solution and Mayer's hematoxylin staining solution. This staining method has stable performance and has been widely used in scientific research and clinical fields. It is recommended as one of the primary methods for amyloid staining. This reagent is for research use only and is not suitable for clinical diagnosis or any other purposes.
A1508467
| Component | 3×50 mL | Storage | | A1508467A | Modified Highman Staining Solution
| 50 mL | RT. Store in the dark. | | A1508467B | Highman Differentiating Solution
| 50 mL | RT | | A1508467C | Mayer's Hematoxylin Staining Solution
| 50 mL | RT. Store in the dark.
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Materials to be Prepared by User
10% neutral formalin, distilled water, graded ethanol, xylene or environment-friendly paraffin removal and clearing solution, neutral balsam
Procedure (for reference only)
1. Perform 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 the sections in Modified Highman Staining Solution for 10-30 min, then discard the residual solution.
4. Differentiate with Highman Differentiating Solution for 1-5 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 5 min.
6. Immerse the sections in Mayer's Hematoxylin Staining Solution for light staining of cell nuclei for 1-2 min or a shorter duration.
7. Rinse with tap water for 10 min.
8. Conduct routine dehydration with graded ethanol, clear the sections with xylene or dewaxing and clearing solution, and mount them with neutral balsam.
Staining Results
Amyloid: Red
Cell Nucleus: Blue
Note: Under a polarized light microscope, amyloid exhibits yellowish-green birefringence.
Modified Highman Staining Solution (Light Staining Effect, Nuclei Stained Blue)
Precautions
1. Ensure thorough dewaxing of sections; otherwise, the staining effect will be affected.
2. Store Highman Differentiating Solution in a sealed container and use it as soon as possible after opening.
3. Prefer immersion staining when using Modified Highman Staining Solution; if drop staining is adopted, place the sections in a moist chamber to prevent solution evaporation.
4. The differentiation step with Highman Differentiating Solution is critical. A 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. Perform dehydration rapidly to avoid decolorization.
6. Due to factors such as tissue specificity or environmental changes, the red color may sometimes appear faint, and the staining time should be appropriately extended in such cases.
7. For your safety and health, wear a lab coat and disposable gloves during operation.
8. Use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
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