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Abstract or Description:

LUXOL FAST BLUE - MODIFIED KLUVER'S - MYELIN SHEATH PURPOSE: Myelin sheath is the white fatty non living material forming an insulating and protective sheath around nerve fibers. PRINCIPLE: Luxol Fast Blue is the alcohol soluble counterpart of the water soluble Alcian Blue. Staining is due to lipoproteins, and the mechanism is one of an acid-base reaction with salt formation; the base of the lipoprotein replaces the base of the dye. CONTROL: Normal cerebral cortex FIXATION: 10% formalin TECHNIQUE: Cut paraffin sections 10μ. Allow to dry in a 37°C oven overnight. EQUIPMENT: Rinse all glassware in DI water, coplin jars, 60°C oven. REAGENTS: Luxol Fast Blue Solution: Luxol fast blue 1.0 gm 95% alcohol 100.0 ml 10% acetic acid 5.0 ml Mix, filter. Solution is stable for 1 year. CAUTION: Flammable liquid, avoid contact and inhalation. 0.05% Lithium Carbonate: Lithium carbonate 0.5 gm Distilled water 1000.0 ml CAUTION: Avoid contact and inhalation. Eosin Y Solution 0.25% Cresyl Echt Violet: Cresyl Echt violet 0.25 gm Distilled water 100.0 ml 10% Acetic acid 1.0 ml Mix, filter. Stable for 2 years CAUTION: Avoid contact and inhalation. SAFETY: Wear gloves, goggles and lab coat. Work in a well ventilated area, avoid contact and inhalation. Acetic acid; moderately toxic on ingestion. Target organ effects on respiratory system on inhalation. Irritant to skin, eyes and respiratory system. Lithium carbonate; irritant to eyes and skin. Alcohol; skin and eye irritant. Ingestion can cause gastrointestinal irritation, nausea, vomiting diarrhea, blindness, death. Excessive inhalation can cause nasal and respiratory irritation. PROCEDURE: 1. Deparaffinize and hydrate to 95% alcohol. 2. Luxol fast blue solution, overnight, 60°C . 3. Rinse in 95% alcohol. 4. Rinse in distilled water. 5. Lithium carbonate solution, 5 seconds. 6. 70% alcohol, two changes, 10 seconds each. 7. Wash in distilled water. 8. Repeat steps 5 - 7 until there is a sharp contrast between the blue of the white-matter and the colorless gray-matter. 9. Rinse slides in 70% alcohol. 10. Eosin, 1 minute. 11. Rinse in distilled water. 12. Cresyl violet, 1 minute. 13. Rinse in distilled water. 14. Dehydrate through 95% and 100% alcohol, clear in xylene, and coverslip. RESULTS: Myelinated fibers blue Neurophils pink Nerve cells purple REFERENCES: Sheehan D, Hrapchak B, Theory and practice of Histotechnology, 2nd ed,1980, pp262-264, Battelle Press, Ohio Crookham,J, Dapson,R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ED, 1991, Anatech

Posted By: Shampa on 3/28/2009 2:34:54 PM
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