Different amino acids carry side chain groups with different chemical properties, some with basic side chains and others with acidic side chains. Proteins composed of these amino acids therefore contain different numbers of basic and acidic groups. These groups cause proteins to carry different net charges in solutions of different pH values. A protein molecule with a net positive charge is referred to as a basic protein (with an isoelectric point biased toward the acidic range); a protein with a net negative charge is referred to as an acidic protein (with an isoelectric point biased toward the acidic range). Taking advantage of the amphoteric nature (acidity and basicity) of proteins and their ability to form salts with acidic and basic dye ions: At pH 2.2 (below the isoelectric point of all proteins), all proteins can be stained by Fast Green. In the range of pH 8.1–8.2, basic proteins are stained by Fast Green.
Basic Fast Green Staining Solution stains by the binding between basic proteins and negatively charged basic dye Fast Green. The most abundant basic protein in cells is histone, which is mainly located in the cell nucleus. Therefore, most of the cell nucleus is stained green after staining. This staining solution is for research use only and is not suitable for clinical diagnosis or other purposes.
Product Components and Storage Conditions:
A1511546
| Component
| 2×50mL
| Storage
|
A1511546A
| Fast Green Staining Solution
| 50mL
| RT. Store in the dark.
|
A1511546B
| Alkaline Buffer
| 50mL
| RT |
Materials Required (User-supplied):
Glass slides, water bath, microscope, pH meter, 70% ethanol, neutral balsam, dilute sodium hydroxide or dilute hydrochloric acid, 5% trichloroacetic acid
Protocol (For Reference Only):
Preparation of Basic Fast Green Staining Solution: Mix Fast Green Staining Solution and Alkaline Buffer in equal proportions. Measure with a pH meter. If the pH is within 8.0–8.5, the solution can be used directly; if outside this range, adjust the pH before use. The prepared staining solution cannot be stored for a long time.
1. Deparaffinize paraffin sections and bring to water.
2. Immerse in 5% trichloroacetic acid, incubate in a water bath at 90 °C for 15 min to extract and remove nucleic acids.
3. Rinse briefly with running water, then wash three times in 70% ethanol, 3 min each time. Blot excess moisture with filter paper.
4. Stain in Basic Fast Green Staining Solution for 20–30 min.
5. Rinse in running water for 5 min, treat with 95% ethanol for 30 s, then absolute ethanol twice, 1 min each time.
6. Clear twice in xylene or deparaffinizing clearing solution, 1 min each. Mount with neutral balsam and examine microscopically.
Staining Results:
Most regions of the cell nucleus: Green
Cytoplasm, nucleoli: Unstained
Precautions:
1. Blood smears or bone marrow smears should be of uniform thickness to avoid affecting the staining result.
2. Fresh whole blood or EDTA-anticoagulated blood is required for blood cell smear staining.
3. If overstained, appropriate decolorization can be performed with methanol or ethanol; restaining is not recommended.
4. pH value has a certain influence on staining. Glass slides should be clean and free from acid or alkali contamination to avoid affecting the staining result.
5. For your safety and health, please wear a lab coat and disposable gloves during operation.
6. This product is for research use only, strictly prohibited for other purposes.
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