Comparison of Hematoxylin and Eosin Staining with and Without Pre Treatment with Marchi ’ S Solution on Nerve Samples for Nerve Degeneration and Regeneration Studies (original) (raw)
Related papers
2017
The study was conducted on four healthy guinea pigs (Cavia porcellus) of either sex in which the nerve was identified and subjected to crush injury with the tip (3mm) of a curved hemostatic forceps. 30 days after the injury nerve samples were collected and subjected to Hematoxylin and Eosin staining with or without pretreatment with Marchi's solution. The routine Hematoxylin and Eosin (H&E) stained all neural elements in various intensities of pink and in purple and the degenerative changes were seen as vacuoles ranging from vacuolated foci-containing eosinophilic material and associated with a distorted cell nucleus to larger, multilocular, linear array of compartmentalized digestion chambers supposed to contain myelin debris .The myelin on the other hand appeared as empty zones in H&E staining. Combining Marchi's and H & E procedures revealed the presence black aggregates/ deposits in the vacuoles and digestion chambers. This method confirmed the presence of degenerated my...
Histochemistry and cell biology, 2011
Peripheral nerves are complex histological structures that can be affected by a variety of conditions with different degree of axonal degeneration and demyelination. For the study of peripheral nerve regeneration in pathology and tissue engineering, it is necessary to evaluate the regeneration, remyelination and extracellular matrix reorganization of the neural tissue. Currently, different histochemical techniques must be used in parallel, and a correlation among their findings should be further performed. In this work, we describe a new histochemical method for myelin and collagen fibers based on luxol fast blue and picrosirius methods, for the evaluation of the morphology, the myelin sheath and the collagen fiber reorganization using a model of peripheral nerve regeneration. Whole brain, normal sciatic nerve and regenerating peripheral nerve samples were fixed in 10% neutral buffered formalin and paraffin-embedded, for the performance of the hematoxylin-eosin stain, the Luxol fast blue method and the new histochemical method for myelin and collagen. The results of this technique revealed that this new histochemical method allowed us to properly evaluate histological patterns, and simultaneously observe the histochemical reaction for myelin sheath and collagen fibers in normal tissue, and during the regeneration process. In conclusion, this new method combines morphological and histochemical properties that allowed us to determine with high accuracy the degree of remyelination and collagen fibers reorganization. For all these reasons, we hypothesize that this new histochemical method could be useful in pathology and tissue engineering.
Journal of Histotechnology, 2007
This review summarizes the function, structure, and chemistry of the myelin sheaths of axons; discusses the mechanisms of several staining techniques using inorganic compounds, dyes, and antibodies; and provides technical instructions for 13 staining methods for normal and degenerating myelin. Myelin has alternating hydrophobic and hydrophilic layers. The former contain the hydrocarbon tails of lipid molecules and hydrophobic segments of transmembrane proteins; the latter contain the anionic heads of polar lipids and also basic proteins. Aqueous fixatives allow some myelin lipids to be retained in paraffin sections. Stains for normal myelin include histochemical reactions that detect choline-containing phospholipids (Baker's chromation-acid hematein) or double bonds (unsaturation) in the fatty acid components of lipids (osmium tetroxide or palladium chloride reduction; the pseudoplasmal reaction and DAB fluorescence, for oxidized double bonds). Traditional fat stains (Sudan dyes) stain normal myelin less intensely than fat or degenerated myelin. Dyes of the luxol fast blue group have colored anions balanced by hydrophobic organic cations. Applied from an organic solvent and differentiated in an aqueous alkali, these dyes are retained in myelin, which is more hydrophilic than fat but more hydrophobic than cytoplasm or collagen. Dye-metal complexes, notably those of ferric iron with hematein ("iron-hematoxylin") or eriochrome cyanine R (also called chromoxane cyanine R, solochrome cyanine R, and Mordant blue 3), are excellent myelin stains for paraffin sections. They probably attach to basic proteins and resist extraction from a relatively hydrophobic environment. Immunohistochemical methods can detect characteristic proteins and lipids of myelin, some of which are different in the central and peripheral nervous systems.
New technique for differential staining of myelinated fibers and nerve cells on paraffin sections
Anat Rec, 1988
A simple, rapid method for the differential staining of myelinated nerve fibers and nerve cell bodies, applicable to sections of central nervous system pieces embedded in paraffin, is described. Experimental material fixed by perfusion with mixed aldehydes or necropsy material fixed in formaldehyde can be used. Constant and homogeneous results are obtained with this technique, and the most important characteristic is the absence of differentiation in either of the steps: staining of myelinated fibers and staining of nerve cell bodies. Sections 15 pm thick were attached to slides, dewaxed, and hydrated. After hydration, sections are mordanted (30 min) in 2.5% iron alum (SO&FeNH4, and rinsed (1 min) in distilled water. Staining is for 180 min in the following solution: 5 ml freshly made 20% alcoholic hematoxylin diluted with 25 ml of distilled water and 25 ml of absolute ethanol to which 10 ml of 1% LiZCO3 is added. The sections are washed in distilled water (5 min) and stained during 5 min in the following solution: 0.2% pyronine, 20% formaldehyde in distilled water. The sections are dehydrated through 96% and absolute ethanol, cleared in eucalyptol, and mounted in Eukitt. Myelinated fibers appear dark blue, whereas nerve cell bodies are stained red and the cell nucleoli dark blue. This procedure provides an adequate contrast for observation and photography.
A Simple Cost Effective Rapid Differential Staining Technique for Myelinated Fibres and Nerve Cells
2012
Nerve cell bodies and nerve fibres are commonly stained by Kluver Barrera method and Margolis method. However few studies have performed for cost effective staining. A simple, cost effective, single stain, rapid method for the differential staining of myelinated nerve fibres and nerve cell bodies applicable to paraffin sections of central nervous system was described. Neutral buffered formalin fixed human cadaver brains and cord were used. Sections of 8 micron thickness of spinal cord, Cerebellum, Pons, Medulla and Mid brain were attached to egg albumin coated slides. Slides were dewaxed and hydrated and brought to water. Slides were stained for two minutes in the following staining solution. 0.25gms of MV10B stain dissolved in 100 ml of autoclaved distilled water and filtered. Stained slides were decolorized in 95% alcohol, dehydrated in absolute alcohol, cleared in xylene and mounted in DPX. Myelinated fibres and nerve cell bodies were stained in varying shades of violet color, wh...
Studies on cultured rat Schwann cells. III. Assays for peripheral myelin proteins
Journal of Neurocytology, 1980
Rabbit antisera to the rat myelin proteins P0 and P1 were used to assay for the presence of these components by both immunochemical and immunofluorescence methods. The antiserum to P0 did not react detectably with polyacrylamide gels containing central myelin, or with Pl and P2 in peripheral myelin; it did react with P0 in peripheral myelin, and in extracts of adult and neonatal sciatic nerve. When reacted with frozen tissue sections using indirect immunofluorescence, it did not stain central myelin but did stain myelin in adult sciatic nerve, the myelinated fibres in cervical sympathetic trunk and occasional areas in neonatal sciatic nerve where Schwann cells had presumably begun to form myelin. Antiserum to basic protein reacted with both of the basic protein bands in central and peripheral myelin, but not P0; P1 and P2 were detectable in adrtlt and neonatal sciatic nerve. In indirect immunofluorescence assays, the antiserum stained both central and peripheral myelin, the few myelinated fibres of sympathetic trunk and myelinating regions of neonatal sciatic nerve.
Isolation, Purification and Verification of Peripheral Nerve Myelin Derived from Bovine Cauda Equina
Journal of Neurology and Neurophysiology, 2012
Background: Animal models of peripheral neuroinflammation are essential to understanding the pathogenesis of, and deducing new treatments for demyelinating polyneuropathies. This paper updates and describes adapted methods for the isolation, purification and verification of bovine peripheral nerve myelin used to reliably induce peripheral neuritis in susceptible mice. Methods: Myelin was isolated via discontinuous sucrose and continuous cesium chloride density gradient ultracentrifugation from frozen adult bovine cauda equina. Negatively stained electron microscopy was employed to confirm myelin structure. Phase contrast and routine light microscopy, gravimetric analyses, gel electrophoresis and lens culinaris fluorescent western blotting, analytical colorimetric assays and indirect immunohistochemistry were performed to analyze whole myelin or its protein and lipid subcomponents. Myelin isolates were used to induce experimental autoimmune neuritis in 8-12 Weeks old female Swedish Jim Lambert/ Jackson mice. Results: Negatively-stained electron microscopy demonstrated the two-dimensional repetitive lamella structure of myelin and a lack of organelle contamination. Lipids accounted for ~72% of the dry mass of bovine peripheral nerve myelin, while ~25% was protein/ proteolipid and <4% insoluble residue based on gravimetric analyses. Glycoproteins accounted for the majority of isolated myelin proteins, with myelin protein zero (~28 KDa) being the most prevalent protein. Phospholipids accounted for ~60% of the total dry weight of bovine peripheral nerve myelin. Surface expression of galactocerebroside and sulfatide, two major lipid subcomponents present in myelin, was detected by indirect fluorescent immunohistochemistry. Bovine peripheral nerve myelin reliably induced a severe macrophagepredominant demyelinating polyneuropathy with associated axonal loss in female mice, consistent with experimental autoimmune neuritis. Conclusions: These updated and adapted methods using readily available resources can be applied to rapidly isolate, purify and verify peripheral nerve myelin from animal sources, and determine its protein and lipid composition prior to use in peripheral neuritis animal models.
Brain Research, 1988
Carbonic anhydrase (CA) and cholinesterase (CE) histochemical staining of rabbit spinal nerve roots and dorsal root ganglia demonstrated that among the reactive myelinated axons, with minor exceptions, sensory axons were CA positive and CE negative whereas motor axons were CA negative and CE positive. The high specificity was achieved by adjusting reaction conditions to stain subpopulations of myelinated axons selectively while leaving 50% or so unstained. Fixation with glutaraldehyde appeared necessary for achieving selectivity. Following sciatic nerve transection, the reciprocal staining pattern persisted in damaged axons and their regenerating processes which formed neuromas within the proximal nerve stump. Within the neuromas, CA-stained sensory processes were elaborated earlier and in greater numbers than CE-stained regenerating motor processes. The present results indicate that histochemical axon typing can be exploited to reveal heterogeneous responses of motor and sensory axons to injury.