Hemosiderin is a hemoglobin-derived pigment that exists as golden yellow or brownish yellow granules. It is named hemosiderin due to its iron content and golden color. When red blood cells are phagocytosed by macrophages, hemoglobin is decomposed into iron-free hemofuscin and iron-containing hemosiderin under the action of lysosomal enzymes. Hemosiderin staining, also known as the Perls Prussian blue reaction, is used to visualize various hemorrhagic lesions in local tissues. It is commonly found in phagocytes and can effectively distinguish hemosiderin from other pigments.
The Prussian Blue Staining Kit (DAB-Enhanced Method) works on the following principle: trivalent iron ions in tissues are dissociated from proteins by dilute hydrochloric acid, and then react with potassium ferrocyanide to form an insoluble blue iron ferrocyanide precipitate, thereby indicating high iron content in tissues. When the iron content in tissues is extremely low, this staining method cannot produce blue precipitate for iron localization. The addition of DAB triggers a redox reaction with the iron ferrocyanide precipitate, forming a brown compound, which enhances the specificity of detection. This method is particularly suitable for tissues with low iron content. This reagent is for research use only and not intended for clinical diagnosis or other purposes.
P1508698
| Component | 5×50 mL | Storage |
| P1508698A | Perls Stain A | 25 mL | RT. Store in the dark. |
| P1508698B | Perls Stain B | 25 mL | RT. Store in the dark. |
| P1508698C | DAB Reagent
| 1 EA | 2-8℃. Store in the dark.
|
| P1508698D | DAB Oxidant
| 1 EA | 2-8℃. Store in the dark. |
| P1508698E | DAB Buffer
| 2×50 mL | RT. |
| P1508698F | Hematoxylin Staining Solution
| 50 mL | RT. Store in the dark. |
| P1508698G | Acid Alcohol Differentiating Solution
| 50 mL | RT. |
Perls Stain Preparation: Mix equal volumes of Perls Stain A (P1508698A) and Perls Stain B (P1508698B). Do not prepare in advance.
DAB Stain Preparation: Add 1 vial of DAB Reagent (P1508698C) into 30 mL of DAB Buffer (P1508698E) and dissolve completely to obtain the DAB solution. Add 1 μL of DAB Oxidant (P1508698D) into 10 mL of DAB Buffer (P1508698E) and mix thoroughly to obtain the DAB oxidation working solution. Immediately before use, mix the DAB solution and DAB oxidation working solution in equal proportions to prepare the DAB Stain.
Self-Prepared Materials
Fixatives: 10% neutral formalin, 4% paraformaldehyde, etc. Graded ethanol, distilled water, xylene, 5% oxalic acid
Operating Procedures (For Reference Only)
Prepare Perls Stain and DAB Stain Before Use:
1. Perls Stain Preparation: Mix equal volumes of Perls Stain A and Perls Stain B. Do not prepare in advance.
2. DAB Stain Preparation: Add 1 vial of DAB reagent into 30 mL of DAB buffer and dissolve completely for later use as the DAB solution; add 1 μL of DAB oxidant into 10 mL of DAB buffer and mix thoroughly as the DAB oxidation working solution; mix the DAB solution and DAB oxidation working solution in equal proportions immediately before use to obtain the DAB Stain.
(1) Staining of Paraffin Sections
1. Fix tissues in 10% neutral formalin fixative, followed by routine dehydration and embedding.
2. Cut sections at a thickness of 4 μm. Deparaffinize to water using routine xylene or paraffin-immersed clearing solution, then rinse with distilled water for 1 min.
3. Immerse sections in Perls Stain (see Note 2) for 15-30 min.
4. Rinse thoroughly with distilled water for 2-5 min.
5. Add DAB Stain dropwise and stain for 5-10 min. Monitor color development under a microscope. Pour off the staining solution, rinse once with DAB buffer, and wash 3 times with distilled water.
6. Stain with hematoxylin for 1-2 min, rinse with tap water, differentiate with acid alcohol differentiating solution for 2-5 s, then rinse with tap water for 5-10 min.
7. Perform routine dehydration, clear with xylene or paraffin-immersed clearing solution, and mount with neutral balsam.
8. Examine under a microscope, then collect and analyze images.
(2) Staining of Frozen Sections
1. No deparaffinization required; directly rinse rapidly with distilled water for 2-3 min.
2. Follow the same staining and mounting steps as for paraffin sections, with appropriately shortened incubation times.
Staining Results
Hemosiderin or trivalent iron: Brownish
Cell nucleus: Light blue
Background or other tissues: Light brown or colorless
Negative Control (Optional)
Take serial sections, deparaffinize to water; incubate in 5% oxalic acid for 2-6 h, stain with Perls Stain, and proceed with the remaining steps as described above; the expected result is negative.
Precautions
1. Ensure thorough deparaffinization of sections. Tissues are usually fixed in 10% neutral formalin; prolonged fixation in ordinary formalin may cause tissue damage. Avoid using acidic fixatives; chromate treatment can also interfere with iron preservation.
2. Keep all containers clean throughout the operation; avoid using iron metal utensils. Use distilled water for rinsing sections and washing containers, as tap water contains iron ions. Adjust the staining time of Perls Stain according to sample conditions.
3. Use the same positive control section for all test sections; selecting an appropriate control is critical. Autopsied lung tissue is an excellent control, as it contains a considerable number of iron-positive macrophages (heart failure cells).
4. Store the prepared DAB solution at -20℃, or at 4℃ for short-term use; the DAB oxidation working solution can be stored at 4℃ for short periods, but should not be left standing for extended times and must be replaced regularly. If staining results are unsatisfactory, increase the amount of oxidant added to the DAB oxidation working solution.
5. During DAB color development, observe the staining under a microscope every 5 min. Stop the reaction when the weak positive signal (light blue) from Prussian blue staining is replaced by DAB to form a brown color, or when the non-blue areas turn brown.
6. For your safety and health, wear a lab coat and disposable gloves during operation.
7. Use the reagents as soon as possible after opening to avoid affecting subsequent experimental results.
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