Staining Nerve Fibers with Silver in Tissue Fixed with Bouin's Fluid

Nerve fibers, in organs fixed with Bouin's fluid, are usually refractive to the Davenport silver technic. The axons, however, can be successfully stained if the sections, on slides, are given a preliminary treatment with concentrated pyridine (1 hour), then a 24-hour bath of ammoniated alcohol (99 cc. 80% alcohol, 1 cc 28% ammonium hydroxide) and an interval in 40% aqueous silver nitrate (6-8 hours) before being immersed in the acidified alcoholic silver solution of Davenport. Following the silvering, reduction and toning of the axons, according to the procedure of Davenport, the surrounding non-nervous tissue elements can be counterstained with a combination of either azocarmine, light green and orange G, or azocarmine, aniline blue and orange G.     

Staining Myelin Sheaths of Optic Nerve Fibers with Osmium Tetroxide Vapor

OsO₄ solution in water, long regarded as the best fixing and staining agent for myelin sheaths, has poor penetrating power. This peculiarity has limited its use to very small pieces of tissue. The vapor from an aqueous solution is known to have a much greater penetrating power for non-neural tissues than the solution itself but nothing has been recorded about its advantages for fixing and staining myelin sheaths of nerve fibers. Difficulties in securing adequate staining of the myelin sheaths in vertebrate optic nerves were overcome largely by the use of the vapor of OsO₄. The technic is carried out as follows: 1) suspend a portion of the nerve above a 2% solution of OsO₄ for 12-24 hours in an air-tight container at room temperature; 2) wash 4-6 hours in distilled water, dehydrate in ethyl alcohol (50% for 2 hours, 70% for 2 hours, and finally 95% overnight), and transfer to n butyl alcohol (2 changes of 2 hours each); 3) embed in paraffin, section, mount and cover in balsam in the customary manner.     

A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

Extensive experimentation with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections IS to 25 μ, frozen sections 25 to 40 μ. Place sections for 24 hours in 50% alcohol to which 1% by volume of NH₄OH has been added. Transfer the sections directly into a 1% aqueous solution of protargol, containing 0.2 to 0.3 g. of electrolytic copper foil which has been coated with a 0.5% solution of celloidin, and allow to stand for 6 to 8 hours at 37° C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24 to 48 hours at 37° C. directly into a pyridinated solution of alcoholic protargol (1.0% aqueous solution protargol, 50 ml.; 95% alcohol, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated copper. Rinse briefly in 50% alcohol and reduce 10 min. in an alkaline hydroquinone reducer (H₃BO₃, 1.4 g.; Na₂SO₃, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroly in water and tone for 10 min. in 0.2% aqueous gold chloride, acidified with acetic acid. Wash in distilled water and reduce for 1 to 3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3 to 5 min. with 5% aqueous Na₂S₂O₃+5H₂O. Wash in water and stain overnight in Einarson's gallocyanin. Wash thoroly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock solution, 20 ml.; distilled water, 30 ml.) of the following stock staining solution: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid, 8 ml.) and stain for 1 hour. Differentiate with 70% and 95% alcohol; pass the sections thru butyl alcohol and cedar oil; mount.     

The Stainability of Nerve Fibers by Protargol With Various Fixatives And Staining Technics

The influence of the commonly used tissue fixing reagents, individually and in various combinations, on subsequent staining by protargol was studied. The reagents used were formalin, formamide, picric acid, acetic acid, paranitrophenol, pyridine and chloral hydrate. Parraffin sections from intestine and peripheral nerve of cat, dog, monkey and rat were stained with protargol after fixation in various experimental mixtures of the fixing reagents. Satisfactory nerve stains of intestine were not obtained with regularity after any one fixing and staining procedure. (Good fixation and staining appeared to be influenced by properties inherent in the tissue itself and showed marked variations from animal to animal even in the same species.)Stains of nerve fibers in peripheral nerve trunks were much more easily obtained than in the intestine where good stains were sporadic and unpredictable. The use of a mixture of 0.5% protargol and 0.1% fast green FCF, is proposed as a silver-dye staining medium.     

Increase of CGRP-Containing Nerve Fibers in the Rat Periodontal Ligament After Luxation

The distribution of calcitonin gene-related peptide (CGRP) was examined in the periodontal ligament (PDL) after experimental luxation injury of the rat first molar tooth. The luxational injury increased the number of CGRP-immunoreactive (IR) nerve fibers. At 3–7 days, numerous CGRP-IR nerve fibers appeared throughout the injured PDL. These nerve fibers terminated as free nerve endings within resorption cavities. Immunohistochemistry for receptor activity modifying protein 1 (RAMP1) also demonstrated that the subunit of CGRP receptor was expressed by periodontal cells adjacent to the alveolar bone in the intact and injured PDL. RAMP1-IR cells were divided into two types; small cells with single nucleus and large cells with 2–6 nuclei. After the luxational injury, both types of RAMP1-IR cells abundantly appeared within resorption cavities. As a result, the treatment increased the number of large RAMP1-IR cells at 3–7 days and small RAMP1-IR cells at 7 days. In addition, a double immunofluorescence analysis demonstrated that CGRP-IR nerve fibers were seen away from RAMP1-IR cells in the intact PDL. After the traumatic injury, however, CGRP-IR nerve fibers appeared in the close vicinity of small and large RAMP1-IR cells at 5–7 days. The morphology and distribution of RAMP1-IR cells suggest that they contain osteoblasts and osteoclasts. By affecting osteoclasts and osteoblasts, CGRP may have effects on bone remodeling in the luxated PDL.     

Staining Nerve Fibers with Methylene Blue. An Evaluation of Variables Used in An Immersion Technic

A study of the effects of osmotic pressure, pH, the presence of dextrose, acetate, pyruvate or lactate, and agents affecting cell permeability during supravital staining by methylene blue was made by means of an immersion technic. Mesentery and intestine of dogs and cats were used. Penetration of the dye was limited to the mesentery and more superficial layers of intestine. Conditions which facilitated the characteristic differentiation of of nerve fibers were: continuous oxygenation of the staining solution, pH about 5.6 stabilized by phosphate buffer, and the presence of small amounts of acetate and lactate. Young animals' tissue stained better than old. Methylene blue was a much more effective staining agent than less completely methylated thionins.     

Staining Paraffin Sections with Protargol 6. Impregnation and Differentiation of Nerve Fibers in Adrenal Glands of Mammals

A series of experiments with protargol staining of nerve fibers in mammalian adrenal glands has yielded the following procedure: Fix-1-2 days in a mixture of formamide (Eastman Kodak Company) 10 cc, chloral hydrate 5 g., and 50% ethyl alcohol 90 cc. Wash, dehydrate and embed in paraffin. Cut sections about 15 and mount on slides. Remove the paraffin and run down to distilled water. Mordant 1-2 days in a 1% aqueous solution of thallous (or lead) nitrate at 56-60°C. Wash thru several changes of distilled water and impregnate in 1% aqueous protargol (Winthrop Chemical Company) at 37-40°C. for 1 to 2 days. Rinse quickly in distilled water and differentiate 7-15 seconds in a 0.1% aqueous solution of oxalic acid. Rinse thru several changes of distilled water for a total time of 0.5 to 1.0 rain. Reduce 3-5 rain, in Bodian's reducer: hydroquinone 1 g., sodium sulfite 5 g., distilled water 100 cc. Wash in running water 3-5 min. and tone 5-10 min. in a 0.2% gold chloride solution. Wash 0.5 min. or more and reduce in a 2% oxalic acid solution to which has been added strong formalin, 1 cc. per 100. (Caution. This last reduction is critical and over-reduction can spoil an otherwise good stain; 15-30 seconds usually suffices, and the sections should show only the beginning of darkening to a purplish or gray color.) Wash, fix in hypo, wash, dehydrate and cover.     

A Differential Staining Method for Elastic Fibers, Collagenic Fibers, and Keratin

A method allowing for the differential presentation of elastic fibers, other connective tissue fibers, epithelial and other types of cytoplasm, and keratin is described. The procedure is based on the affinity of orcein for elastic fibers, of anilin blue for collagenic material, and of orange G for keratin. Bouin-fixed, tissue-mat embedded sections are stained in Pinkus' acid orcein for 1 1/2 hours and rinsed in distilled water. The sections are differentiated in 50% alcohol containing 1% hydrochloric acid, washed in tap and then in distilled water. The sections are next transferred for I to 2 minutes to the anilin blue, orange G, phosphomolybdic acid combination known as solution No. 2 of Mallory's connective tissue stain, diluted 1:1 with distilled water. They are then rinsed in distilled water, quickly passed into 95% alcohol, and dehydrated in absolute alcohol containing some orange G, after which they are cleared and mounted. Within less than two hours sections may be stained and mounted with the following results: elastic fibers — red; collagenic fibers — blue; muscle fibers — yellow; keratin — orange.     

Thiazin Dyes in Supravital Staining of nerve Fibers

Supravital staining by thiazins of segments of small intestine and mesentery of young dogs was studied with reference to specificity for nervous tissue. Attempts to secure a purer form of methylene blue by alumina adsorption and alcohol elution of the commercial, medicinal dye yielded a product which appeared to be structurally different from the original dye. The treated dye had absorption maxima from 620 to 655 mμ in contrast with 665 for the untreated. Small nerve bundles were stained by the treated dye after 2 to 4 hours of immersion, but staining was always incomplete. Staining by untreated methylene blue was compared with that by the leucobase, thionol, methylene green, toluidine blue, new methylene blue and the azures. It was concluded that the specificity for nerve fibers resides mainly in the =N(CH₃)₂Cl radical, although some specificity appears to be effected by the methyl groups on the trivalent nitrogen, since azure A (dimethyl) and azure C (mono-methyl) stained weakly, but thionin did not. Methylene green showed some specificity but stained weakly. The leucobase was less active than the reoxidized dye obtained from it.     

Staining Formalin-Fixed Nerve Tissue with Mercuric Nitrate

Experiments were made to test the impregnating effect of Hg(NO₃)₂ on nervous tissue that had been fixed and chromated with solutions of known pH. Brains of cats, kittens, rats and mice were fixed by the pulsating-perfusion method of Haushalter and Bertram (1955), after first washing out the blood with saline-acacia solution, at pH 7.0, then followed by a 10% formol-saline-acacia fixative of the same pH. The removed brains were sliced to 3 mm thickness and further fixed 1-2 days in 10% formalin whose pH was also adjusted to 7.0. Chromation with acidified ZnCrO₄ at pH 3.1 for 1 day followed by impregnation for 2 days in a saturated solution of Hg(NO₃)₂ at pH 5.5-6.0 effected the staining. Dehydration, paraffin embedding and sectioning completed the process. Some moderately successful stains were made with mercuric salts with no chromation, but it was found that fixation at pH 7.0-7.2 followed by chromation at pH 3.1, and later by impregnation in Hg(NO₃)₂ at pH 5.8-6.0 was optimum for best staining of nerve cells for their processes. The advantages of the technique are: (1) selective staining of nerve cells, especially the axonic details; and (2) a relatively short time needed for its completion.     

Microwave Assisted Staining of Nerve and Muscle Biopsy Tissue

This paper reports a technique using microwaves to assist penetration of stains into biopsy sections of muscle and peripheral nerve. The technique results in more consistent and reliable staining of tissue sections for examination by light microscopy.     

Microwave Assisted Staining of Nerve and Muscle Biopsy Tissue

This paper reports a technique using microwaves to assist penetration of stains into biopsy sections of muscle and peripheral nerve. The technique results in more consistent and reliable staining of tissue sections for examination by light microscopy.     

Visualizing Peripheral Nerve Regeneration by Whole Mount Staining

Peripheral nerve trauma triggers a well characterised sequence of events both proximal and distal to the site of injury. Axons distal to the injury degenerate, Schwann cells convert to a repair supportive phenotype and macrophages enter the nerve to clear myelin and axonal debris. Following these events, axons must regrow through the distal part of the nerve, re-innervate and finally are re-myelinated by Schwann cells. For nerve crush injuries (axonotmesis), in which the integrity of the nerve is maintained, repair may be relatively effective whereas for nerve transection (neurotmesis) repair will likely be very poor as few axons may be able to cross between the two parts of the severed nerve, across the newly generated nerve bridge, to enter the distal stump and regenerate. Analysing axon growth and the cell-cell interactions that occur following both nerve crush and cut injuries has largely been carried out by staining sections of nerve tissue, but this has the obvious disadvantage that it is not possible to follow the paths of regenerating axons in three dimensions within the nerve trunk or nerve bridge. To try and solve this problem, we describe the development and use of a novel whole mount staining protocol that allows the analysis of axonal regeneration, Schwann cell-axon interaction and re-vascularisation of the repairing nerve following nerve cut and crush injuries.     

Diffusion of Ions in Myelinated Nerve Fibers

The diffusion of ions towards or away from the inner side of the nodal membrane in preparations, the cut ends of which are placed in various media, was investigated. The ion concentration changes were calculated by numerical solution of the unidimensional electrodiffusion equation under a variety of media compositions, axoplasmic diffusion coefficients, and internal anionic compositions. The potassium and cesium ion diffusion along the axon towards the node was determined experimentally by two different electrophysiological methods. On the basis of comparison between the experimental data and the computational predictions the axoplasmic potassium ion diffusion coefficient was determined to be almost equal to that in free aqueous solution, while that of cesium ion was close to one half of that in aqueous solution. Utilizing the values of diffusion parameters thus determined, we solved the electrodiffusion equation for a number of common experimental procedures. We found that in short fibers, cut 0.1-0.2 cm at each side of the node, the concentration approached values close to the new steady-state values within 5-30 min. In long fibers (over 1 cm long) steady-state concentrations were obtained only after a few hours. Under some conditions the internal concentrations transiently overshot the steady-state values. The diffusion potentials generated in the system were also evaluated. The ion concentration changes and generation of diffusion potential cannot be prevented by using side pools with cation content identical to that of the axoplasm.     

Staining Normal and Experimental Motor Nerve Terminals with Tetrazolium Salts

Nitroblue tetrazolium (NBT) has been used to stain motor nerve terminals and unmyelinated axons in vertebrate skeletal muscle, but undesirable background connective tissue coloration resulted. This procedure was improved by separation of the tetrazolium salt's binding from its subsequent reduction. By uncoupling the binding and reduction steps it was possible (1) to improve nerve terminal staining by using tetranitroblue tetrazolium (TNBT), (2) to counterstain and postfix in osmium tetroxide and (3) to enhance the overall tissue preservation. The separate binding and reduction procedure is compatible with postsynaptic acetylcholinesterase staining. Experimentally manipulated and diseased preparations can be successfully stained, and the requirements for optimal staining in each case are described.