10.1 Nerve Tissue & Myelin: Luxol Fast Blue, Bielschowsky & Silver Methods
Key Takeaways
- Luxol fast blue is a copper phthalocyanine dye that binds the lipoprotein of myelin sheaths, staining intact white matter blue-green and leaving demyelinated plaques pale.
- Luxol fast blue is a regressive method differentiated in 0.05 percent lithium carbonate followed by 70 percent alcohol, checked microscopically until grey matter is colorless and white matter remains blue.
- Cresyl echt violet counterstains Nissl substance purple after Luxol fast blue, producing the standard LFB-CV combination for demyelinating disease.
- Bielschowsky ammoniacal silver impregnates axons, neurofibrillary tangles, and neuritic plaques black and is the classic histochemical method for Alzheimer disease neuropathology.
- Neuropathology silver methods require acid-cleaned glassware, deionized water, and non-metallic forceps because trace metal or chloride reduces silver into background precipitate.
10.1 Nerve Tissue & Myelin: Luxol Fast Blue, Bielschowsky & Silver Methods
ASCP HT Exam Focus: Nerve is an explicitly named procedure under Staining in the ASCP BOC content outline, and the Summary of Stains lists Luxol fast blue (LFB/cresyl echt violet) and Bielschowsky by name. Items typically pair the target (myelin versus axon versus Nissl substance) with the correct method.
Central nervous system tissue contains three distinct structures that routine H&E cannot separate: the myelin sheath (a lipoprotein spiral), the axon (a fine argyrophil neurofilament process), and the Nissl substance (ribosome-rich rough endoplasmic reticulum in the neuronal cell body). Neurohistology uses a different technique for each.
Preparing Neural Tissue
- Prolonged fixation. Whole brains are suspended by the basilar artery in a large volume of 10 percent neutral buffered formalin for 10 to 14 days before gross cutting; unfixed brain is too soft to section reproducibly.
- Section thickness. Myelin and silver methods are typically cut at 8 to 15 micrometers rather than the routine 4 micrometers, because myelin sheaths and axonal processes must be followed through the plane of section.
- Adhesive slides. The long, hot, alkaline, and alcoholic baths of these methods lift tissue readily; use positively charged or silane-coated slides.
Luxol Fast Blue: Myelin
Luxol fast blue (LFB) is a copper phthalocyanine dye supplied as an alcohol-soluble salt. The dye's base exchanges with the base of the lipoprotein in the myelin sheath, forming an alcohol-insoluble colored complex. Because myelin is preserved as lipoprotein rather than free lipid, LFB works on routine formalin-fixed paraffin sections, unlike the lysochromes of section 9.4.
Method outline
- Bring sections to 95 percent alcohol (not to water - the dye is alcoholic).
- Incubate in 0.1 percent Luxol fast blue in 95 percent alcohol with acetic acid at 56 degrees C to 60 degrees C overnight, or at 60 degrees C for 2 to 4 hours.
- Rinse off excess dye in 95 percent alcohol, then in distilled water.
- Differentiate in 0.05 percent lithium carbonate for seconds only, then complete differentiation in 70 percent alcohol, checking microscopically.
- Endpoint: grey matter is colorless to pale, white matter is sharply blue to blue-green. Repeat the lithium carbonate and alcohol cycle if grey matter still retains dye.
- Counterstain, dehydrate, clear, and mount resinously.
Counterstain combinations
| Combination | Counterstain result | Typical use |
|---|---|---|
| LFB - cresyl echt violet (LFB-CV) | Nissl substance and nuclei purple-violet | Demyelinating disease, neuronal loss |
| LFB-PAS | Basement membrane, glycogen, fungal wall magenta | Storage disease, infection |
| LFB-H&E | Standard nuclear and cytoplasmic detail | General neuropathology survey |
Interpretation trap: a pale, sharply demarcated area on an LFB slide is demyelination (multiple sclerosis plaque, leukodystrophy, progressive multifocal leukoencephalopathy). Diffuse weak blue across the whole section is under-differentiation reversed too far - a technical failure, not a diagnosis. Always read the control block.
Silver Methods for Axons and Neurofibrillary Pathology
Axons, neurofibrils, and the abnormal filamentous aggregates of neurodegenerative disease are argyrophil: they bind silver ions but require an external reducing agent to convert them to visible metallic silver.
Bielschowsky (and the modified Bielschowsky)
- Impregnate hydrated sections in 20 percent silver nitrate in the dark at 37 degrees C.
- Transfer to ammoniacal silver (silver nitrate to which ammonium hydroxide is added dropwise until the precipitate just redissolves).
- Develop in dilute formalin, which reduces the bound silver to black metallic silver; watch microscopically.
- Tone in 0.2 percent gold chloride, then fix in 5 percent sodium thiosulfate and counterstain if desired.
Results: axons, neurofibrillary tangles, and the dystrophic neurites of neuritic (senile) plaques are black on a yellow-brown to grey background. The modified Bielschowsky remains a standard method in Alzheimer disease staging protocols.
Related argyrophil methods
- Bodian: uses protargol (silver proteinate) with metallic copper in the impregnating bath and hydroquinone reduction; nerve fibers and neurofibrils black. Widely used for peripheral nerve and myenteric plexus.
- Sevier-Munger: a faster modification of Bodian, also used for neuroendocrine granules.
- Holzer and phosphotungstic acid hematoxylin (PTAH): demonstrate glial fibers in gliosis; PTAH is covered in section 10.4.
- Cresyl violet alone (Nissl stain): a simple basic thiazine that colors ribosomal RNA of Nissl substance violet, used for neuronal counting and cytoarchitecture.
Immunohistochemistry has replaced many of these for specific targets - neurofilament for axons, GFAP for astrocytes, tau, beta-amyloid, and alpha-synuclein for degenerative inclusions - but the histochemical silvers remain on the ASCP content outline and in routine neuropathology panels.
Silver Handling Rules
The same discipline described for reticulin and methenamine silver applies here and is a frequent exam item:
- Use acid-cleaned, silver-dedicated glassware; detergent film nucleates precipitation.
- Use deionized or distilled water only; tap water chloride throws down silver chloride as a black granular veil.
- Handle slides with plastic, Teflon, or glass forceps. Stainless steel reduces silver on contact and blackens the slide edge.
- Add ammonium hydroxide to silver nitrate drop by drop only until the brown precipitate just clears. Excess ammonia makes the solution too weak to impregnate; a stored ammoniacal silver solution can form explosive silver nitride and must be made fresh and discarded the same day.
- Collect silver waste for reclamation; silver is an EPA-regulated heavy metal.
A Luxol fast blue stain on a brain section from a patient with suspected multiple sclerosis shows uniformly pale blue white matter with residual blue in the grey matter as well. The control block from the same run shows the same pattern. What is the correct interpretation and corrective action?
A neuropathologist requires demonstration of neurofibrillary tangles and the dystrophic neurites of neuritic plaques in a hippocampal section. Which histochemical method is the classic choice, and what reagent converts the bound silver to a visible black deposit?
During a silver impregnation for axons, a histotechnician prepares ammoniacal silver, uses it, and stores the leftover solution in a stoppered bottle on the bench for use the following week. Which hazard does this practice create?