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A General-Purpose Method of Silver Staining by A. PETERS (Frovijhe Department of Zoology, University of Bristol)

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A General-Purpose Method of Silver Staining by A. PETERS (Frovijhe Department of Zoology, University of Bristol) 323 A General-Purpose Method of Silver Staining By A. PETERS (Frovijhe Department of Zoology, University of Bristol) SUMMARY A method of silver staining for paraffin sections has been described. Sections should be fixed in either Nonidez fixative, 4% formaldehyde, or 4% formaldehyde saturated with mercuric chloride. The sections are impregnated for 16 hours in 1/20,000 silver nitrate at pH 8 or 9 and developed in a glycine physical developer after the reducible silver has been removed with a 2% solution of sodium sulphite. The effect of pH on impregnation has been described. A spectrum of staining was obtained in which nerve fibres began to stain appreciably at pH 7, cell nuclei at pH 8, cell cytoplasm at pH 9, and connective tissue at higher pH values. Therefore, impreg- nation should be carried out at pH 8 to obtain a good staining of nerve fibres and at pH 9 if some staining of cell bodies is also required. N recent years, a number of methods for the silver staining of paraffin sections of nervous tissue have been described. Probably the most impor- Itant have been those of Holmes (1947), Romanes (1950), and Samuel (19536). Although these methods vary in detail, they have the common factor that impregnation is carried out at a controlled pH in a dilute solution of a silver salt. Holmes and Samuel used silver nitrate and Romanes used silver chloride. While Holmes and Romanes employed a hydroquinone-sulphite developer, which reduced the silver taken up by the sections during impregnation, Samuel removed the reducible silver with a sodium sulphite solution and developed in a physical developer. Thus, in Samuel's method the silver which is reduced to produce the final staining picture is derived from the developing, solution and not from the silver combined with the section during impreg- nation (see Peters, 19556). By this means the development process can be controlled to a greater extent than is possible with the chemical developers, such as hydroquinone-sulphite. The staining method to be described in this paper has been evolved as a result of a series of experiments on the mechanism of silver staining (Peters, 1955 a, b, c, and d). Some observations on fixation and the effect of pH of the impregnating solution will also be considered, because these two factors play an important part in the production of the final staining picture. Fixation Rowe and Hill (1948) considered the use of various fixatives before stain- ing by the method of Holmes (1947). They concluded that Susa, chloral hydrate, and mercury-formol fixatives gave the best results, but they pointed out that chloral hydrate fixatives produce severe shrinkage of the tissue. A series of simple fixing agents, including picric acid, potassium dichrom- ate, chromic acid, 70% alcohol, and formalin were tested. The results showed [Quarterly Journal of Microscopical Science, Vol. 96, part 3, pp. 323-328, 1955.] 324 Peters—A General-purpose Method of Silver Staining that of this group, only formalin- and alcohol-fixed tissue gave consistently good staining results. However, while alcohol-fixed material gave rise to a good staining picture, the fixation was poor. As a result of these and other experiments, it was concluded that the best staining was produced after fixa- tion in Nonidez fixative (25 g. of chloral hydrate in 100 ml. of 50% alcohol (Nonidez, 1939)) in 4% formaldehyde, and in 4% formaldehyde saturated with mercuric chloride. While the Nonidez fixative produced some shrinkage of the tissue, it had the advantage that, after staining, the nerve elements stood out clearly against the other tissue elements. Formalin produced better fixation than the chloral hydrate, but the staining of the nerve fibres was not so well differentiated. The addition of mercuric chloride to the formalin has the effect of increasing the depth of staining of the nerve fibres and suppressing the staining of the background, so that the contrast was improved. When mercury-formol is used, the precipitate which is formed during fixation must be removed from the tissue with 2% iodine in 70% alcohol before impregnation in the silver solution. The excess iodine should not be removed from the sections with sodium thiosulphate, because, unless the thiosulphate is completely removed, it interferes with the silver staining. The pH of the impregnating solution The effect of pH on the staining of the cerebellar region of the rat's brain was determined over the pH range 4-5 to 11-2. Sections were impregnated in a 1/20,000 solution of a silver salt for 16 hours at 37° C. A solution of silver nitrate was buffered at pH 4-5 and 5-6 with sodium acetate / acetic acid buffer and at pH 7-0, 8-o, and 90 with borax / boric acid buffer. At pH 9-4, 9-9, 10-3, 10-7, and 11-2 a 1/20,000 solution of the silver diammine complex was em- ployed and the pH of the solution was controlled by the addition of either sodium carbonate or ammonia. (Silver nitrate could not be used at these high pH values because of the formation of silver hydroxide.) The sections were either developed in 1% hydroquinone / 10% sodium sulphite or in the glycine physical developer. After development in the hydroquinone-sulphite developer, there was an increase in the density of staining from pH 4-5 to 9-0. When the pH was controlled by the addition of sodium carbonate to the diammine complex, staining increased from pH 9-4 to n-2, but when the pH was adjusted with ammonia, there was a fall-off in the intensity of staining with an increase in the pH. The addition of ammonia probably reduced the ionization of the diammine complex and thereby suppressed the release of free silver ions available for staining. Thus, the solution was stabilized, and any tendency for it to reduce or combine with proteins of the nervous tissue was retarded (Peters, 1955a). At higher pH, when the greatest concentration of ammonia was added to the solution, there was the lowest concentration of silver ions available for staining. As long as the free silver ion concentration in the impregnating solution was Peters—A General-purpose Method of Silver Staining 325 constant, the intensity of staining, on development with hydroquinone-sul- phite, increased with the pH. As the pH was raised, there was a tendency for the deposited silver to become coarse, although this factor was not important until about pH 10-7. Development with the glycine physical developer showed that with a con- stant silver ion concentration in the impregnating solution, the formation of TABLE I The effect of pH on silver staining Intensity of staining Nerve Connective pH fibres Nuclei Cytoplasm tissue Comments 4-S-S-6 + + + + Staining too light to show details. 7'o + + + -f- Fibres begin to stain. Staining still very light. 80 -j- -!- Fibres contrasted + + + + + against light back- ground. 90-99 + + + + + + + -f Quite good staining of all nerve elements. 103 + + + + + + Fibre staining rather coarse. 107 + + + + + J L- Cell nuclei appear as outlines. Staining generally coarse. 112 + + + + T- Few details visible; + + + staining very coarse and homogeneous. + faint; ++ distinct; deep. silver nuclei increased with an increase in the pH value of impregnation (see Peters, 1955a). The effect of pH on the staining of the various nerve elements and con- nective tissue is shown in table 1. An interesting point brought out by this table is the spectrum of nerve-element staining which is obtained as the pH is raised (Silver, 1942). Thus, fibres and nuclei begin to stain at the lower pH values, while the cytoplasm only begins to stain appreciably at pH 9-0. Moreover, the specific staining of the cytoplasm persists to a higher pH than that of the nerve fibres and cell nuclei. Some of the specificity is lost at higher pH as a result of a staining of the connective tissue. It can be seen from table 1 that the best pH for impregnation is 8-o to 9-0, because over this range the deepest staining of the nerve fibres and nuclei is obtained. In general, the staining of the nerve fibres is deepest at pH 8-o and there is little staining of the other tissue elements at this pH. At pH 9-0 the staining of the cell nuclei is deeper, but there is some reduction in the intensity of staining of nerve fibres. However, a more complete general 326 Peters—A General-purpose Method of Silver Staining staining of the nerve elements results at pH 9-0 than at pH 8-o. At all other pH values the staining is either too light or too unspecific. In their methods, Holmes (1947) impregnated at pH 8-5, Romanes (1950) at pH 9-0, and Samuel (19536) at pH 678. A possible explanation for the spectrum of staining lies in the physical state of the proteins at the different !pH values. Silver (1942) suggested that the spectrum effect was caused by a variation in charge on the cellular components, so that parts of the cell stain at different pH values and have an 'optimum magnitude of charge to absorb the nascent colloidal silver'. Although it is doubtful if Silver's theory of staining is generally correct (see Holmes, 1947, and Samuel, 1953a), the pH effect may well be due to a difference in charge on the proteins at different pH values. A further possibility is that the sites of formation of the silver nuclei change with pH; this would lead to the developed silver being deposited at different sites over the pH range. Such a change in the sites of formation of nuclei could be attributed to a change in the redox potential of the cell proteins with the pH value (see Peters, 1955a).
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