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 yellow color. When red blood cells are phagocytosed by macrophages, hemoglobin is decomposed into iron-free hematoidin and iron-containing hemosiderin under the action of lysosomal enzymes. The Perls Prussian blue reaction, also known as hemosiderin staining, produces a blue color after treatment with potassium ferrocyanide and dilute acid. This reaction is commonly observed in phagocytes or interstitium and is primarily used to demonstrate ferric iron salts. In addition, the Lillie method is also an excellent and highly specific technique for detecting ferric iron.
L1508457
| Component | 2×50 mL | Storage | | L1508457A | Lillie Ferric Iron Solution A
| 25 mL | RT. Store in the dark.
| | L1508457B | Lillie Ferric Iron Solution B
| 25 mL | RT. Store in the dark.
| | L1508457C | Nuclear Fast Red Staining Solution
| 50 mL | RT. Store in the dark. |
Preparation before use: Mix equal volumes of Solution A and Solution B to prepare Lillie Stain. Do not prepare the stain in advance.
|
Operating Procedure
1. Fix the tissue in 10% neutral formalin or other alkaline fixatives, followed by routine dehydration and embedding.
2. Cut sections at a thickness of 4 μm and perform routine dewaxing to water.
3. Rinse the sections with distilled water for 1 minute.
4. Immerse the sections in Lillie Stain (refer to Precaution 4) and incubate for 25-30 minutes.
5. Rinse thoroughly with distilled water for 2-5 minutes.
6. Transfer to Nuclear Fast Red Staining Solution and lightly stain the cell nuclei for 5-10 minutes.
7. Rinse with distilled water for 1-5 seconds.
8. Perform routine dehydration and clearing, then mount the sections with neutral balsam.
Staining Results
Ferric iron: Deep Prussian blue
Cell nuclei and other tissues: Red
Negative control (optional): Take a corresponding control section, dewax to water, and the result should be negative.
Precautions
1. This staining method is suitable for paraffin sections, frozen sections, and resin sections. Ensure thorough dewaxing of the sections.
2. Tissues are usually fixed in 10% neutral formalin. Prolonged fixation in regular formalin may cause tissue damage. Avoid using acidic fixatives; chromate treatment can also interfere with iron preservation.
3. Use clean containers throughout the operation and avoid iron metal utensils. Rinse the sections and containers with distilled water, as tap water may contain iron impurities.
4. Adjust the incubation time with Lillie Stain according to the characteristics of the sample.
5. Use the same positive control section for all test sections; selecting an appropriate control is crucial. Autopsied lung tissue, which contains a substantial number of iron-positive macrophages (heart failure cells), is an excellent control.
6. Replace the graded ethanol solutions regularly with fresh ones.
7. For staining frozen sections and cells, optimize the experimental conditions according to the specific sample type.
8. For your safety and health, wear a lab coat and disposable gloves during operation.
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