Lipids are a general term for neutral fats, lipids, and their derivatives. Their common physical property is insolubility in water and solubility in organic solvents (e.g., ethanol, ether, etc.). There are two main types of fats in the human body: Storage fats (e.g., neutral fats), mainly distributed in the subcutaneous tissue, kidneys, pancreas, and other parts; Structural fats (e.g., lipids including phospholipids, glycolipids, cholesterol, etc.), mainly distributed inside cells. Phospholipids are composed of glycerol, fatty acids, and nitrogen-containing bases, serving as the main structural components of cell membranes and intracellular membranes. They are abundant in tissues such as the brain, nerve tissue, bone marrow, and liver. Phospholipids can be divided into three types: lecithin, cephalin, and sphingomyelin. Common methods for demonstrating phospholipids include the acidic hematoxylin method and pyridine extraction method.
Phospholipid Staining Solution (Acidic Hematoxylin Method), also known as Baker’s Phospholipid Stain, uses calcium formaldehyde to fix tissues. Calcium ions prevent phospholipids from dissolving into the fixative solution. After treatment with Baker’s mordant, phospholipids bind to metal ions to form insoluble phospholipid chelates, which then stain into blue-black complexes with acidic hematoxylin. Phospholipids and sphingomyelin appear blue-black under staining.Note: This reagent is for research use only. Not for clinical diagnosis or other purposes.
P1508519
| Component | 3×100 mL | Storage | | P1508519A | Baker Mordant
| 100 mL | RT | | P1508519B | Acidic Hematoxylin Staining Solution
| 100 mL | RT. Store in the dark. | | P1508519C | Baker Differentiating Solution
| 100 mL | RT |
|
Materials to Be Prepared by User
1. Distilled water, graded ethanol, glycerin gelatin, calcium formaldehyde fixative
2. Incubator or water bath
Operating Procedures (For Reference Only)
1. Fix tissues in calcium formaldehyde fixative for 16 hours (overnight).
2. Immerse the fixed tissues in Baker’s mordant in a 60℃ incubator for 24 hours, followed by rinsing under running water for 16 hours (overnight).
3. Section the tissues using a cryostat or semiconductor refrigeration microtome at a thickness of 8 μm.
4. Wash the sections with distilled water and air-dry.
5. Re-immerse the sections in Baker’s mordant in a 60℃ incubator for 1 hour, rinse briefly under running water, and wash again with distilled water.
6. Stain the sections in acidic hematoxylin stain in a 37℃ incubator for 2-5 hours, then rinse under running water.
7. Differentiate the sections in Baker’s differentiating solution in a 37℃ incubator for 0.5-2 hours, rinse under running water for 10 minutes, and wash with distilled water.
8. Mount the sections with glycerin gelatin.
Staining Results
Phospholipids, Sphingomyelin, Nucleoprotein: Blue–Black
Cytoplasm Grayish: Yellow
Negative Control (Optional)
1. Prepare a phospholipid extraction reagent: chloroform:methanol (2:1) or pyridine.
2. Treat consecutive tissue sections with the above extraction reagent at room temperature for 1–2 hours.
3. Perform phospholipid staining using the same procedure; the result should be negative.
Precautions
1. This staining method may produce false positives. If necessary, treat sections with a lipid extraction reagent and perform a negative control.
2. Acidic hematoxylin stain is for single use only. Do not reuse it.
3. The differentiating solution can be reused until its yellow color fades significantly, then discard it.
4. For your safety and health, wear a lab coat and disposable gloves during operation.
5. Use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.
ZJ26F0332879