Safranin O-Fast Green Staining Solution (for Plant Tissues) mainly consists of Safranin O Staining Solution and Fast Green Staining Solution. After staining sections of root, stem, leaf and other tissues, safranin stains the nucleus and lignified cell walls bright red, cutinized cell walls transparent pink, and suberized walls reddish-brown; fast green stains the cytoplasm and cellulose-containing cell walls bluish-green. Differentiation is critical in safranin O-fast green staining: over-differentiation easily leads to no staining of sections, while under-differentiation results in overly intense staining. This reagent is for research use only and not for clinical diagnosis or therapy.
Protocol
1. Specimen Preparation: Fix the specimen and prepare paraffin sections.
2. Section Mounting: Cut the material into thin sections, mount the sections on glass slides, and heat to spread the sections. Slides must be clean. First apply adhesive to the slide, then place the section floating on the adhesive, and set the slide on a slide warmer to flatten the section until no wrinkles appear. Incubate in an incubator at 30–40 ℃ for approximately 1 h. Adhesives used include gelatin, Mayer’s albumin, Land solution, etc.
3. Dewaxing: Xylene → 50% Xylene + 50% Ethanol → 100% Ethanol → 95% Ethanol → 85% Ethanol → 70% Ethanol → 50% Ethanol → 30% Ethanol → Water. Each step requires 5–10 min.
4. Stain with Safranin O Staining Solution for 2–12 h.
5. Differentiate with Acid Ethanol Differentiating Solution for several seconds, then wash with water to remove excess stain.
6. Decolorization: 35% Ethanol → 50% Ethanol → 70% Ethanol → 80% Ethanol. Each step requires 30–60 s.
7. Rinse briefly with distilled water.
8. Counterstain with Fast Green Staining Solution for 1 min.
9. Dehydrate through 95% Ethanol and two changes of absolute Ethanol, 30 s–1 min each. Clear in 50% Xylene + 50% Ethanol for 5 min, then two changes of Xylene, 5 min each.
10. Mount with resin and examine microscopically promptly.
Staining Results
Lignified, suberized and cutinized cell walls
| Red
|
Cellulose-containing cell walls
| Bluish-green |
Notes
- After safranin staining, decolorization in 50% Ethanol requires practical adjustment. Insufficient decolorization will hinder fast green staining; excessive decolorization will result in faint red staining or even complete green staining.
- Fast green is a rapidly staining dye; staining time should not be too long, otherwise the safranin color will be faded.
- Staining time is not absolute and often varies with material type and section thickness.
- For your safety and health, please wear a lab coat and disposable gloves during operation.
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