Solarbio Prussian Blue Iron Stain Kit(with Neutral Red Solution)

Introduction

• Hemosiderin Characteristics:
  • Derived from hemoglobin breakdown, it appears golden-yellow or brownish-yellow due to iron content.
  • Formed when macrophages engulf red blood cells and break down hemoglobin into iron-free orange blood and iron-containing hemosiderin.
• Perls Prussian Blue Reaction:
  • Also known as hemosiderin staining.
  • Produces a blue color when treated with potassium ferrocyanide and dilute acid.
  • Commonly seen in phagocytes’ interstitium, displaying ferric iron salts.
  • Classical histochemical reaction, sensitive, and excellent for displaying ferric iron in tissues.
  • The principle involves separating ferric iron from protein using potassium ferrocyanide solution and hydrochloric acid, forming an insoluble Prussian blue compound.
  • Stable ferrocyanide of ferric iron allows re-dyeing with red dye after reaction.
• Perls Stain Usage:
  • Commonly used to display hemorrhagic lesions, especially in phagocytes.
  • Helps determine hemosiderin deposition and distinguish it from other pigments.
  • The staining solution is stable, long-lasting without precipitation, and suitable for a wide range of applications.
  • Can be re-dyed, making it versatile for different staining techniques.

Kit Components

Before use, mix equal parts of A1 and A-2 to form Perls Stain. It is not suitable to prepare in advance.

Self Provided Materials

10% neutral formalin fixative, Series of ethanol, Distilled water, 4% paraformaldehyde

Protocol (for reference only)

(1) For paraffin section staining

1. Fix the tissue in 10% natural formalin fixative, dehydrate, and embed.
2. Cut sections 4µm thick, dewax to distilled water, and rinse for 1 minute.
3. Soak the section in Perls Stain for 15-30 minutes. Rinse fully in distilled water for 2-5 minutes.
4. Lightly stain the nucleus with a Neutral Red Solution for 15-30 seconds. Rinse in tap water for 1-5 seconds.
5. Conventionally dehydrate, transparent, and seal with resinene.

(2) For frozen section staining

1. Without dewaxing, rinse directly and quickly with distilled water for 2-3 minutes.
2. Follow the other steps as paraffin sections.

(3) For cultured cell staining

1. Fix in 4% paraformaldehyde for 10-20 minutes.
2. Rinse in tap water twice for 2 minutes each time.
3. The steps of staining, dehydration, transparency, and sealing are the same as paraffin section steps. Adjust time accordingly.

Result

• Hemosiderin or Ferric Iron: Blue
• Nucleus and other Tissues: Red

Negative Control

Take the same adjacent section, dewax to water. After incubation in 5% oxalic acid for 2-6 hours, follow the same procedure as above. The result should be negative.

Note

1. Ensure clean section dewaxing.
2. Use 10% neutral formalin for tissue fixation. Long-term fixation with common formalin can damage tissues. Avoid acid fixatives, as chromate treatment hinders iron preservation.
3. Keep containers clean and avoid using metal iron products. Use distilled water for washing sections and containers to prevent iron contamination.
4. Adjust Perls Stain dyeing time according to the sample.
5. Use the same positive control section for all sections. Autopsy lung tissue is a good control, containing many iron-positive macrophages (heart failure cells).
6. Replace ethanol series frequently.
7. For frozen section and cell staining, explore experimental conditions based on specific conditions. Wear experimental clothes and disposable gloves for health and safety.