Viral hepatitis is an infectious disease characterized by degeneration and necrosis of liver parenchymal cells, which is caused by hepatitis viruses. Hepatitis B virus (HBV) is a globally prevalent hepatitis virus, and hepatitis B surface antigen (HBsAg) is present in the cytoplasm of hepatocytes. At present, immunohistochemical methods can be used to detect HBsAg and hepatitis B core antigen (HBcAg) of HBV. The methods for detecting HBsAg include Shikata’s orcein method, aldehyde-fuchsin method, Victoria blue method, etc.
Aladdin Hepatitis B Virus Staining Solution (Shikata’s Orcein Method) features simple operation and high specificity, but the staining effect may vary with different batches of raw materials. The potential mechanism is that orcein binds to the disulfide bonds of cystine in HBsAg, thereby producing color development. This reagent is intended for research use only and is not suitable for clinical diagnosis or any other purposes.
Product Components and Storage Conditions:
H1510444
| Component
| 3×50mL
| Storage
|
H1510444A
| Shikata Oxidant
| 25mL
| RT. Store in the dark.
|
H1510444B
| Shikata Acid Solution
| 25mL
| RT
|
H1510444C
| Shikata Bleaching Solution
| 50mL
| RT
|
H1510444D
| Acidic Orcein Staining Solution
| 50mL
| 4°C. Store in the dark. |
Prior to use, mix equal volumes of Solution A and Solution B to prepare the Shikata Acidified Solution. The solution should be prepared fresh and used immediately.
Materials Required (Self-prepared):
10% formalin fixative, distilled water, graded ethanol solutions
Operating Procedures (For Reference Only):
1. Fix the tissue in 10% neutral formalin fixative, followed by routine dehydration and embedding.
2. Cut into sections with a thickness of 4 μm, then perform routine dewaxing to water using xylene or dewaxing clearing solution.
3. Immerse the sections in Shikata acidified solution for oxidation for 5 minutes, then rinse briefly with water.
4. Transfer the sections into Shikata bleaching solution for bleaching for 2 minutes to remove the stain from the acidified solution, followed by rinsing under running water for 2 minutes.
5. Rinse the sections slightly in 70% ethanol. Then place them in acidic orcein staining solution, cover the container, and stain for 5 hours or longer.
6. Clear the sections with xylene or dewaxing clearing solution, and mount the sections with neutral balsam.
Staining Results:
HBsAg-positive Substances
| Reddish-brown to dark brown
|
Background
| Pale red of varying degrees
|
Precautions:
1. Shikata acidified solution must be prepared fresh and used immediately. Prolonged storage after mixing will reduce its oxidizing capacity.
2. Bring the acidic orcein staining solution to room temperature before use. The staining solution can be reused several times.
3. After 5 hours of staining, the sections can be removed, rinsed briefly in 70% ethanol, and then washed with distilled water for microscopic examination. If the staining is faint, rinse the sections in 70% ethanol and then restain them in acidic orcein staining solution; overnight staining is permissible if necessary.
4. Lipofuscin may also yield a positive reaction with orcein staining. The distinguishing features are as follows: lipofuscin stains darker and appears as uniformly sized fine granules in the cytoplasm; whereas HBsAg stains lighter and presents as fine granular deposits.
5. For your safety and health, wear a lab coat and disposable gloves during the operation.
6. Please use the reagents as soon as possible after opening to avoid compromising subsequent experimental results.
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