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, impregnation 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 impregnation (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 staining 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 fixation 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 employed 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-sulphite, 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 constant 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 background. 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 connective 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). Thus, the number of silver nuclei formed at a particular site would depend on the value of the redox potential at that site.

Method of staining (1) Fix blocks of tissue in either Nonidez fixative (1939) or 4% formaldehyde or 4% formaldehyde saturated with mercuric chloride. (2) Mount paraffin sections on slides with albumen, dewax, and take to water. (If mercury-formalin has been used for fixation, remove the precipitate from the sections with 2% iodine in 70% alcohol.) (3) Impregnate sections in the following solution in an incubator for 16 hours: 1 ml. of 1% silver nitrate, 180 ml. of distilled water, and 20 ml. of o-i M boric acid / borax buffer at pH 8 or 9. The standard buffer solution is made by mixing solutions of o-i M boric acid and 01 M borax until the required pH, as indicated by a glass electrode, is attained. Impregnate at 37° C. for material fixed in chloral hydrate and at 56° C. or 370 C. for formalin and mercury-formalin material. (4) Transfer sections to 2% sodium sulphite for 5 minutes to remove the reducible silver (Samuel, 1953a). . (5) Wash in several changes of distilled water. (6) Develop the sections in the following glycine-containing physical developer until the required depth of staining is attained. Sections should be examined at intervals during development. The usual time is 2-5 minutes. glycine - 1-25 g.\

INa,SO,(anhyd.) *5 * 20 ml. 5% gelatine (powdered B.P.) 25 ml. distilled water 225 ml./ o-i M citric acid / sodium citrate buffer at pH 6-3 . 20 ml. 1 % silver nitrate solution ..... 1 ml. Peters—A General-purpose Method of Silver Staining 327 (7) Wash in running tap water for 10 minutes. (8) Dehydrate, take through absolute alcohol to xylene, and mount in Canada balsam. The stock solution of the developer is quite stable. To prepare this the glycine and sodium sulphite are dissolved, by warming, in about 100 ml. of distilled water and the warm gelatine solution is added immediately. The volume is then made up to 250 ml. The optimum pH for development may vary slightly with the sample of gelatine; initial tests should be carried out with citrate buffers over the range pH 6-o to 6-5. However, once the pH value has been determined for any particular sample of gelatine, no further tests are necessary. The citrate buffer controls the pH value at the site of development; on either side of the optimum pH the silver deposition is rather coarse. In general, no toning is necessary, because the fibres show up black against a green background and therefore give a good contrast. The reducible silver may be removed with a citrate buffer at pH 3-2, but the sodium sulphite is more convenient to prepare. Unless the reducible silver is removed, the initial development is rapid and tends to be somewhat unspecific. The temperature of impregnation varies with the fixative and the type of tissue. For example, formol-fixed rat cerebellum produced the best staining picture at 56° C. and formol-fixed human cerebellum at 370 C. On the other hand, material fixed with chloral hydrate never produced specific staining after impregnation at 560 C. Whether the sections are impregnated at pH 8 or 9 depends on the nerve elements that are required in the final staining picture. As has already been pointed out, at pH 8 the staining of the nerve fibres is deep and that of the non-nervous elements is light, while at pH 9, although more background is stained, the nerve-cell bodies stain more intensely. Deeper staining may be produced by treating the sections with 20% silver nitrate for 1 hour before impregnation (Holmes, 1947). In the present method this step is generally unnecessary. It is recommended that embryonic tissues should be fixed in Nonidez fixative and dehydrated in the Lang series of alcohols (Lang, 1937). A less perfect staining of nerve fibres may be achieved by developing the impregnated sections in one of the following solutions. Here, development follows step 3 in the above scheme. (1) hydroquinone. . . . ig. Na2SO3 (anhydrous) . . 10 g. distilled water . . 100 ml. Warm the solution to 200 C. before use. (2) chloroquinol . . . . 1 g. Na2SO3 (anhydrous) . . 4 g. distilled water . . 100 ml. Use at room temperature. 328 Peters—A General-purpose Method of Silver Staining While the basic method of staining in the above scheme produces good results, the modifications have been listed because a rigid method of staining cannot be expected to produce the best possible results with all tissues and fixatives. Thus, initial trials should be carried out to determine the pH and temperature of impregnation which gives the best staining with the sections available. This method has been used successfully with fish, amphibian, and mam- malian tissues, including brain, spinal cord, sciatic nerve, sympathetic gang- lia, muscle end-plates, and embryonic material.

Note.—The glycine mentioned in these papers is the compound called by that name in photography; that is to say, ^>-hydroxyphenylglycine.

I wish to express my sincere thanks to Professor J. E. Harris for his con- tinued interest and advice during the course of this work. The work was carried out during the tenure of a postgraduate research grant in the Univer- sity and later a maintenance grant from the Department of Scientific and Industrial Research.

REFERENCES HOLMES, W., 1947. In Recent advances in clinical pathology. London (Churchill). LANG, A. G., 1937. 'The use of M-butyl alcohol in the paraffin method.' Stain Tech., 12, 113- NONIDEZ, J. F., 1939. 'Studies on the innervation of the heart. I. Distribution of the cardiac nerves with special reference to the identification of the sympathetic and parasympathetic postganglionics.' Amer. J. Anat., 65, 361. PETERS, A., 1955a. 'Experiments on the mechanism of silver staining. I. Impregnation.' Quart. J. micr. Sci., 96, 84. • '9556. 'Experiments on the mechanism of silver staining. II. Development.' Ibid., 96, 103. J955C- 'Experiments on the mechanism of silver staining. III. Quantitative studies.' Ibid., 96, 301. 1955a1. 'Experiments on the mechanism of silver staining. IV. Electron microscope studies.' Ibid., 96, 317. ROMANES, G. J., 1950. 'The staining of nerve fibres in paraffin sections with silver.' J. Anat. Lond., 84, 268. ROWE, M.( and HILL, R. G., 1948. 'The effects of various fixatives on the staining, by Holmes' method, on axons in the central and peripheral nervous systems.' Bull. Inst. med. Lab. Tech., 14, No. 4. SAMUEL, E. P., 1953a. 'The mechanism of silver staining.' J. Anat. Lond., 87, 278. I953&- 'Towards controllable silver staining.' Anat. Rec, 116, 511. SILVER,,M. L., 1942. 'Colloidal factors controlling silver staining.' Ibid., 82, 507.