Golgi potassium-dichromate silver-nitrate impregnation

Summary Golgi preparations were made by consecutive treatment of formalin-fixed brain and liver with potassium dichromate and silver nitrate. Impregnated tissue dissected from thin slices of the blocks were studied by X-ray powder diffraction methods, in a diffractometer and a Guinier camera. Such tissue proved to contain crystalline silver chromate, Ag₂CrO₄, both while still in the silver nitrate solution and after dehydration in ethanol and clearing in xylene and xylene-Dammar resin. No other compounds containing chromium or silver were detectable. Formalin-fixed tissue merely treated with silver nitrate contained silver chloride, but in impregnated tissue the amount was too scarce to be visible. Hence, silver chloride was no integral part of the Golgi precipitate.A number of mostly ethereal oils traditionally used for clearing histological sections, did not cause the appearance of metallic silver in detectable amount in the Golgi preparations. However, after treatment with clove oil and creosote metallic silver was detected in the tissue.This study was supported by U.S. P.H. S. Grant NS 07998. This aid is gratefully acknowledged.We are indebted to Miss I. Madsen and Mrs. K. Sörensen for technical assistance.     

A Method for Visualizing the Acrosome by Light Microscopy

A silver staining technique applied to squash preparations of material previously fixed in 3:1 ethanol: acetic acid produces differential staining of the acrosomal region of spermatids during spermiogenesis in orthopteroid species. The method includes treatment with saline sodium citrate solution for 15 min at 60 C, and staining with 50% aqueous silver nitrate adjusted to pH 2.9 with formic acid.     

A method for visualizing the acrosome by light microscopy

A silver staining technique applied to squash preparations of material previously fixed in 3:1 ethanol:acetic acid produces differential staining of the acrosomal region of spermatids during spermiogenesis in orthopteroid species. The method includes treatment with saline sodium citrate solution for 15 min at 60 C, and staining with 50% aqueous silver nitrate adjusted to pH 2.9 with formic acid.     

An Improved Silver Stain for Developing Nervous Tissue

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.l% formol as developed agent, 25% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

An improved silver stain for developing nervous tissue.

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.1% formol as reducing agent, 2.5% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

The Bielschowsky Staining Technic a Study of the Factors Influencing Its Specificity for Nerve FIBERS

By comparing results obtained with adult mammalian tissue from introducing variables into each separate step in block-staining by the Bielschowsky silver method, the following conclusions were reached:

1. No specific means for inhibiting the staining of connective tissue and still permitting complete staining of nerve fibers was found, but the avoidance of overstaining was very helpful toward such differentiation.
2. Overstaining could be corrected by reducing the concentration of the silver nitrate bath or by adding an excess of ammonia to the ammoniated silver bath.
3. Staining of fine fibers was favored by adding acetic acid to the formaldehyde used for fixation or by adding pyridin to the silver nitrate bath.
4. Addition of protein-precipitating organic acids (trichloracetic or sulfosalicylic) to the fixative was disadvantageous.
5. Prolonged fixation favored an increase in intensity of the stain. Four days' time was sufficient.
6. Extraction of lipids with ammoniated alcohol gave results similar to those obtained after extraction with pyridin, but the stain was lighter.
7. Ammoniated silver carbonate without excess ammonia had an action similar to ammoniated silver hydroxide with excess ammonia.
8. An excess of ammonia in the ammoniated silver solution (Ag 0.1 N) was tolerated, without apparent impairment of nerve-fiber staining, up to 6 M NH₃, altho the use of more than 3 M excess (2 cc. concentrated ammonia water added to 100 cc. of balanced ammoniated silver hydroxide solution) seemed unnecessary.
9. Impregnation with 1.7% (0.1 N) silver nitrate solution was quite satisfactory and variations in the concentrations of this bath suggested that the practical limits of concentrations that would be generally satisfactory lay between 0.3 and 3.0%.
10. The writers' experiences agreed with Agduhr's relative to the advantage of washing in 2.5% acetic acid between the ammoniated silver bath and formaldehyde reduction.

     

Differentiation of Myofibrillae, Reticular and Collagenous Fibrils in Vertebrates

A procedure for the differentiation of the mesenchymal derivatives, myofibrillae, reticular and collagenous fibers is presented. Formol-Zenker fixation (5-12 hours) is followed by the washing, iodinization, dehydration and paraffin embedding steps routine for that fixative with the following modifications. Zirkle's butyl alcohol series is used for dehydration and infiltration with paraffin as well as in the alcohol slide series. Embedding paraffin used is Parawax plus 8-10% bayberry wax. Tissue-exposed surface of paraffin block is soaked in water overnight before cutting serial sections at 3-5μ. Sections are mounted using the dilute albumen method, and the slides, thoroughly dried at 37^oC. overnight, are left at 60^o for 10 minutes to melt the paraffin of the sections. Before staining, the sections are given a preliminary treatment with potassium permanganate and oxalic acid. For reticular staining a 10% silver nitrate bath is succeeded by an ammoniacal silver carbonate solution followed by reduction in 1% neutral formalin, toning in gold chloride and fixing in sodium thiosulphate. Myofibrillae, the sacroplasmic limiting membrane and other sarcous elements are stained by Heidenhain's azocarmine solution, adult tissues at room temperature and fetal tissues at 50 ^oC. Differentiation in phosphotungstic acid is followed by the staining of collagenous fibers. For adult tissue, light green SF (C.C.) is used and for fetal tissue, fast green FCF (C.C). A discussion of the preparation of ammoniacal silver solutions is included. Both stock and used solutions of ammoniacal silver have been in use by the author for over a period of two years.     

Demonstration Of Golgi Zones By Three Technics For Carbohydrates

The carbohydrate of the Golgi apparatus of several organs of rats, rabbits, and frogs was selected as the principal test material for the behavior of three different technics: 1) periodic acid with colored fuchsin; 2) “direct” chromic acid piperazine silver; 3) periodic acid with leucofuchsin.

Parallel sections of organs in which positive reactions were observed, were treated before staining with a series of reagents to characterize them as glycoprotein.

The results obtained by the three technics under any constant set of conditions were essentially identical in all cases. It is concluded that discrepancies that may have been noted up to now are due to several factors, probably the most important being the tissue's physiological status and the influence of fixation. The study shows that HIO₄, -fuchsinl and chromic acid silver methods are, at least empirically, as valid as HIO₄, -leucofuchsin technics.

Considering the differences in the oxidative mechanism of chromic and periodic acids and other data, the possibility of two different chemical pathways leading to the same final result is discussed.

It has been found that colored fuchsin, as well as its leuco form, can be used in the histochemical demonstration of aldehydes after periodic acid treatment (Arzac, 1948). In a later report (Amc, 1950), a series of reactions were obtained with colored fuchsin which differed in several ways from the results of others using Hotchkiss' method. For example, Gersh (1949) reported the presence of probable glycoproteic granules in the Golgi apparatus of rabbit and guinea pig's intestine. Leblond (1950) also found positive Golgi reactions in different cells of male excretory ducts and in other organs of the rat. Such reactions had not been observed with the colored fuchsin technic in any of the two above-mentioned occasions.

Since the latter investigators used different fixatives, which might have caused the discrepancies, the experiment described below was undertaken to study: (a) the influence of fixation on the final re-actions elicited by HI04-fuchsin (colored and leuco-form) and chromic acid piperazine silver methods; (b) the results obtained in the demonstration of Golgi zones of several rat's, rabbit's and frog's organs by these methods.     

Mechanism of Prophylaxis by Silver Compounds against Infection of Burns

To clarify tthe mechanism by which local application of silver compounds protects burns against infection, an ion-specific electrode was used to measùre the concentration of silver ions in solutions. By this method it was shown that in burn dressings silver ions were reduced to a very low level by precipitation as silver chloride. The antibacterial effect was found to depend on the availability of silver ions from solution in contact with precipitate. Between 10^-5 and 10^-6 molar silver nitrate solution in water was rapidly bactericidal. The minimal amount of silver nitrate causing inhibition of respiration of skin in tissue culture was about 25 times the minimal concentration of silver nitrate that inhibited growth of Pseudomonas aeruginosa.     

A silver carbonate method for cell counts of neurons and glial elements on paraffin embedded brain tissue.

A modification of the Del Rio-Hortega method for the demonstration of central nervous system elements is presented. This silver impregnation technique is particularly useful for the classification of cell types for quantitative differential cell counts. Formalin fixed paraffin sections are immersed in formol-ammonium bromide for 1 1/2 hours; this solution is an excellent mordant for various silver nitrate stains. The samples are stained for 20 to 60 minutes in a silver carbonate solution (25 ml of 25% silver nitrate combined with 200 ml of 5% sodium carbonate) and then reduced in a 1% formaldehyde solution to which 20 drops of acetic acid have been added. Finally, the slides are fixed in sodium thiosulfate, rinsed in tap water, dehydrated, cleared, and mounted. This procedure will enable this investigator to identify neurons, oligodendroglia, and astrocytes on the basis of their nuclear staining as well as to demonstrate the laminae of brain tissue since the method allows differentiation of cell layers and fiber tracts.     

Immunofluorescent Labelling of K-Papovavirus Antigens in Glycol Methacrylate Embedded Material: A Method for Studying Infected Cell Populations by Fluorescence Microscopy and Histological Staining of Adjacent Sections

Trypsin and protease V (pronase) were studied for their ability to enhance immuno-fluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 μm sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Chloride distribution in the CA1 region of newborn and adult hippocampus by light microscopic histochemistry

GABA, the main inhibitory neurotransmitter in the central nervous system, exerts its effect by rendering the postsynaptic GABAA receptors permeable to chloride ions. Thus, depolarizing or excitatory effects of GABA, experienced in early postnatal life or in certain regions and/or conditions of the adult brain, is thought to be associated with a reversed transmembrane chloride gradient. However, there is only limited direct information about the correlation of the actual excitatory versus inhibitory effects of GABA and the local chloride distribution. Precipitation of chloride with silver is a potential way to immobilize and visualize chloride ions in biological tissue. We examined the applicability of light microscopic histochemistry, based on trapping tissue chloride with silver ions during freeze-substitution or aldehyde fixation, to visualize the chloride distribution in hippocampal slices. The freeze-substitution procedure yielded better chloride retention while with aldehyde fixation tissue preservation was more appropriate. Both methods were qualitative only, had limited applicability to the superficial 20-30 microns of slices, but were able to demonstrate a reduced extracellular-to-intracellular chloride gradient in the CA1 pyramidal neurons of the newborn hippocampus as compared to adult animals. In the 4-aminopyridine model of epilepsy, redistribution of chloride from extracellular to intracellular space could also be demonstrated.     

Immunofluorescent labelling of K-papovavirus antigens in glycol methacrylate embedded material: a method for studying infected cell populations by fluorescence microscopy and histological staining of adjacent sections

Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Acid Versus Neutral Formalin Solution as a Neurological Fixative

The fixing action of 10% formalin solution alone and with formic, acetic, propionic, butyric, lactic, monochloracetic, dichloracetic, or trichloracetic acid was studied by means of stains with silver, osmic acid and cresyl violet. The following conclusions were reached:

1. In general, better fixation and staining was obtained with acid than without.

2. Less difference was seen in comparing one acid with another than was expected before the experiments were made.

3. Propionic, butyric, and dichloracetic showed no promise of having practical value.

4. Formic and monochloracetic acids as modifiers gave superior stains with osmic acid, while silver and cresyl violet stains of the same material were about equal to those made from formalin-acetic fixed material.

5. Lactic acid caused somewhat more distortion of tissue elements than the others but was compatible with good staining.

6. Acetic acid was most effective in concentrations of 3 to 5% while the stronger acids such as formic, monochloracetic, lactic and trichloracetic were effective in concentrations of 0.5 to 1%.     

Suppression of connective tissue impregnation in a silver technique for demonstrating nerve fibers.

A tissue pretreatment is introduced which effectively suppresses the silver impregnation of connective tissue and nonspecific 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% alcohol 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 beginning 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 utilizing fresh frozen 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 is suppressed by the use of mercuric chloride in the fixative and that the background suppression is related to the short fixation time with formolsublimate.     

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.     

On the specificity of the alcoholic,acidic silver nitrate reagent for the histochemical localization of ascorbic acid

Summary The defects besetting the histochemical localization of ascorbic acid were removed in the modified method described here by the simultaneous fixation of the experimental material and its reaction with silver nitrate by the use of alcoholic, acidic silver nitrate reagent in the dark at 0–3°C for 24 hours or longer at pH 2–2.5.The fixatives like acetic acid and alcohol of the reagent ensure quick penetration of AgNO₃ for fixation of ascorbic acid in situ before sectioning. It has been experimentally established that none of the other reductants react with AgNO₃ at the pH and the temperature mentioned.The sections were devitaminized by treatment with 6–10% formaline for 3–4 hours to serve as a control.     

Glutaraldehyde-Ammoniacal Silver Carbonate-Formaldehyde Staining of Histones of Mitotic Chromosomes

Cells from monolayer culture of Chinese hamster line Don were treated by Colcemid (0.1 μg/ml) for 2 hr, trypsinized and spun; resuspended in 0.5% sodium citrate solution for 10 min, respun, and then resuspended in a small volume of the supernatant. Slide preparations were made by smearing, followed by air drying for 1 min at room temperature. They were fixed and stained by the following sequence: 2.5% glutaraldehyde in Millonig's buffer, 30 min; distilled water, 6 min, 5 changes; ammoniacal silver at 18-26 C, 10 sec; distilled water, 30 min, 5 changes; 2.5% formalin, 2 min; and distilled water, 3 changes during 15 min. Staining solution: add 225 ml of 5% Na₂CO₃ to 75 ml of 10% AgNO₃, then add concentrated NH₄OH slowly, drop by drop, until the solution is transparent. Finally add 300 ml of dstilled water. Cells treated with cold 0.25 N HCl before fixation were not stained. Sequence modifications show that chromatin does not reduce silver by itself. This method stains the sites of high histone concentrations in mitotic chromosomes of cytogenetic preparations.     

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.     

A Urea Silver Nitrate Method for Nerve Fibers and Nerve Endings

A silver nitrate stain for nerve fibers and endings applicable to paraffin sections on the slide utilizes the properties of urea to accelerate the procedure and improve the specificity of the stain. After removal of the paraffin the sections are run through absolute, 95% and 80% alcohol and placed for 60-90 minutes at 50-60°C. in: 1% aqueous silver nitrate, 100 ml.; urea, 20-30 g.; 1g. mercuric cyanide and 1 g. picric acid in 100 ml. of distilled water, 1-3 drops. After the silver bath they are rinsed quickly in 2 changes of distilled water and reduced for 3-5 minutes at 25-30°C. in: water, 100 ml.; sodium sulfite, anhydrous, 10g.; hydroquinone, 1-2g.; urea, 20-30g. They are then washed thoroughly in 4-5 changes of distilled water, passed through graded alcohols into 80% alcohol and examined under the microscope. If nerve fibers are not distinct, the sections are returned to the same urea-silver-nitrate bath for 10-15 minutes, rinsed, reduced, washed and dehydrated as before. This process may be repeated until staining is adequate; then they are dehydrated, cleared, and mounted.

Nerve fibers show a color range from brown to black; nerve cells from yellow to brown; and the background, depending on the type of tissue and its fixation, from yellow to light brown.