Staining Nerve Fibers After Sublimate-Acetic and After Bouin'S Fluid

A silver staining method for paraffin sections of material fixed in HgCl₂, sat. aq., with 5% acetic acid is as follows. Process the sections through the usual sequence of reagents, and including I-KI in 70% alcohol, thiosulfate (5% aq.), washing and back to 70% alcohol containing 5% of NH₄OH (conc. aq.). After 3 minutes in the ammoniated alcohol, wash through tap water and 2 changes of distilled water and silver 5-10 minutes at 25°C. in 15% AgNO₃ aq. to which 0.02 ml. of pyridine per 100 ml. has been added. Blot the slide, but not the section and do not rinse. Reduce at 45°C. in 0.1% pyrogallol in 55% alcohol, then rinse in 55% alcohol and wash in water. The remainder of the process consists of gold toning, intensifying in oxalic acid, fixing in 5% Na₂S₂O₃, washing, dehydrating, clearing and covering. When the specimen contains much smooth muscle, the I-KI solution is acidified before use by adding 2 ml. of 1N nitric acid per 100 ml., and the sections treated for 3 minutes instead of the usual 2 minutes. Formalin should not be added to sublimate-acetic, but specimens that do not contain strongly argyrophilic nonneural tissue may be fixed in formalin or, preferably, Bouin's fluid. Sections of tissue after the latter type of fixation will not require the I-KI and thiosulfate but can go from 95% alcohol to the ammoniated alcohol. The advantages of fixing in HgCl₂-acetic acid are suppression of the staining of connective tissue and intensifying the staining of nerve fibers.     

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.     

Suppression of Connective Tissue Impregnation in a Silver Technique for Demonstrating Nerve Fibers

A tissue pretreatment technique is introduced which effectively suppresses the silver impregnation of connective tissue and nompecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcobol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginnins with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilidng fresh hrozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation H suppressed by the use of mercuric chloride in the fixative and that the background supprgsion is related to the short fixation time with formol-sublimate.     

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.     

Block Staining of Nervous Tissue with Silver IV. Embryos

Procedures having enhanced reliability over older methods for both Bielschowsky and Cajal types of stain are described.

Fixation of embryos in a solution containing 4% formaldehyde and 0.5% trichloracetic acid greatly improved the staining of neural elements by Bielschowsky's method.

Among the variations of Cajal's type of staining tried, a modification of Ranson's pyridin-silver method gave the most complete staining of neurofibrillar elements. Washing for 0.5 to 1 hour after silver impregnation and shortening of the reduction time from 24 to 4 hours corrected the tendency of the method to overstain.     

应用微波辐射促进组织块浸银染色

目的：探索微波辐射对组织块浸银染色各步骤的作用及微波在其中的应用方法。方法：在组织块浸银染色过程中,对组织块的固定,浸银,还原等步骤进行不同强度的微波辐射处理。结果：微波辐射明显促进了浸银,还原作用,应用适宜强度的微波辐射染色效果比常规染色有更多的优点。结论：恰当地应用微波辐射缩短组织块浸银染色时程,切实可行。     

Complete Staining of Nerve Fiber and Myoneural Junctions with Acetylthiocholine and Silver

We describe a combined stain for simultaneous demonstration of the preterminal axons and cholinesterase activity at myoneural junctions of mammalian muscles. This technique employs acetylthiocholine iodide as the substrate for cholinesterase activity and silver nitrate impregnation of preterminal axons. The procedure is rapid, simple and Uses fresh muscles. Intramuscular nerves, preterminal axons and myoneural junctions are stained simultaneously brown or black with minimal background staining of connective tissue and muscle fibers.     

Complete Staining of Nerve Fiber and Myoneural Junctions with Acetylthiocholine and Silver

We describe a combined stain for simultaneous demonstration of the preterminal axons and cholinesterase activity at myoneural junctions of mammalian muscles. This technique employs acetylthiocholine iodide as the substrate for cholinesterase activity and silver nitrate impregnation of preterminal axons. The procedure is rapid, simple and Uses fresh muscles. Intramuscular nerves, preterminal axons and myoneural junctions are stained simultaneously brown or black with minimal background staining of connective tissue and muscle fibers.     

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.     

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.     

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.     

A New Method of Silver Impregnation for Nerve Cells and Fibers

Fragments of tissue, immediately after death, are fixed in Debaisieux's modification of the Duboscq-Brazil picro-aceticformol fluid, and treated as follows: Hydrate by soaking 2-6 hr. in distilled water with 30 drops of cone. NH₄OH per 100 cc. Freeze and cut sections about 25μ in thickness. Bleach sections about 15 min. in ammoniacal water (52 drops cone. NH₄OH per 100 cc. water). Transfer to 20% AgNO₃ solution and heat at 45° C. till light brown. Add cone. NH₄OH drop by drop till the Ag precipitates and then redisolves into an opalescent solution. Pour solution and sections into a little distilled water and transfer sections quickly to formaldehyde solution (3 cc. formalin to 100 cc. water). Dip sections in distilled water and transfer to 1% aqueous gold chloride till deep blue. Place for about 10 minutes in 5% aqueous sodium thiosulfate solution for fixing and clearing. Wash thoroly in tap water, dehydrate and mount. Special directions are given for applying this technic to delicate material such as insects, and for use with serial sections.     

Block Staining of Nervous Tissue with Silver: III. Pericellular end-bulbs or boutons

Fixation in 20 to 40% pyridin or in 10% chloral hydrate followed by 10 to 40% pyridin gave the most consistent staining of pericellular structures in the spinal cord of cat. Chloral hydrate perfusion and soaking followed by ammoniated alcohol (Hoff's application of Cajal's method) was uniformly successful only when pyrogallol instead of hydroquinone was used as a reducing agent. Perfusion of the animal with chloral hydrate gave a rather questionable degree of improvement over fixation by simple soaking. The difficulty in selecting a routine procedure as the “best” became apparent when no single experimental variation was outstandingly superior in all animals.     

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.     

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.