Silver Staining Methods

SILVER STAINING METHODS (see also Impregnation) compete in microscopy with chromogenic techniques. Two categories can be distinguished: see: old methods applied to fresh living tissues and newer ones applied to tissues with preliminary processing. An example of the first category is the treatment of a single-layer epithelium with a solution of silver nitrate. Reduction occurs here in the intervals between cells, resulting in black, wavy lines in the form of a network and a negative image of the cells themselves. For instance, take a membranous organ (mesentery, pericardium) from a recently killed animal, stretch it on a cork or wax plate, secure it with cactus needles, and immerse it in a 1% or weaker solution of silver nitrate. Within 1–10 minutes, a milky turbidity appears; then the preparation is rinsed with distilled water and placed in a white, preferably porcelain, cup filled with distilled water in the light. A browning occurs—a sign of reduction; after that, it is transferred to distilled water (to which 2 grains of table salt have been added beforehand) 70% and 90% alcohol. Warm gelatin solution or the latter injected in a mixture with silver nitrate solution can be introduced into blood vessels, lungs, serous cavities, and similar places. In the cornea, intermediate cells (cells of the anterior epithelium, which have been removed by steam action beforehand) sometimes give a positive, sometimes a negative image, being totally impregnated together with their processes. On myelinated fibers, silver is deposited in the nodes of Ranvier, and the nearest parts of the axons are also captured, resulting in Ranvier's crosses and Frommann's transverse lines on the axon. In some organs, silver is introduced deep into the tissue by a Pravaz syringe. In addition to pure silver nitrate solution (0.1–0.75%), mixtures are sometimes used, for example, 1% Arg. nitr. and 2% Ac. osmic. (Kolosov). Picric and nitric acids are also added to it. Furthermore, in addition to Arg. nitr., acetates, picrates, lactates of silver, protargol, and others are used. In 1877, Goyer (Norway) first introduced ammoniacal silver. In addition to the action of light for reduction, formalin, hydroquinone, etc., can be used, and for removing excesses—hyposulfite. The second category of silver staining includes methods with preliminary tissue processing. Very many of them succeed after universal formalin fixation, especially in combination with cutting tissues on a freezing microtome. Some of them selectively reveal various structural details, for example, the Golgi apparatus, while others, in their detail, can replace chromogenic methods such as hematoxylin-eosin. In this case, staining can be supplemented with post-staining (scarlet-red, rottin, picrofuchsin, etc.). For formalin fixation, 10–14–20% solution is taken; duration—from 3 days. 96% alcohol, pure or with ammonia (0.5–1%), is also used; also formalin with alcohol (60%) in a ratio of 1:9; acetone, acetone half with formalin, and acetone with nitric acid (10%). After formalin fixation, pyridine is sometimes used, or pyridine is added to the silver salt solution. According to Schulze, formalin-fixed tissues are cleaned of it in a solution of caustic soda before silver staining. Furthermore, before silver staining, in some methods, substances similar in action to pro-herbs in chromogenic methods are introduced: arsenious acid, uraninite, tannin, iron alum, ammonium bromide, bromural, etc. Arg. nitr. is used in 1–10% solutions; argentamine, colloidal silver, carbonate, fluoride, lactate, and silver nitrite are also used. Ammoniacal silver (Goyer, Fajersztajn, Belynovsky) proved especially valuable. Tissues are processed in these methods both in pieces and in frozen sections. The change in the colloidal medium is of very great importance. For this purpose, gelatin embedding (12.5%, 20% and 24%) is used, or the Arg. nitr. solution is mixed with a warm gelatin solution (1 to 1 000); gum arabic is also used. There are the most diverse techniques for silver staining. For example, sections lie in Arg. nitr. for a day, then are quickly passed through ammoniacal silver after light rinsing in distilled water and then quickly subjected to reduction (Belynovsky); or sections are quickly passed through Arg. nitr., then formalin, ammoniacal silver, ammoniacal water, etc. (Gross-Belschovsky). In addition to 20% formalin for reduction, grape sugar, hydroquinone, pyrogallol, adurol, gallic acid, etc., are used. Very black sections can be weakened with a 0.05%–0.2% solution of potassium cyanide or potassium permanganate. Subsequent gold plating of sections (a few drops of 1% chloroauric acid in a portion of water) and 5% hyposulfite are very useful, but not always mandatory. The most important part ends with reduction. The most important methods of the second category. For nerve cells with processes—the Golgi and Cox method (see Golgi method); for neurofibrils and nerve fibers—the Ramon y Cajal method (see Ramon y Cajal staining methods) and Belynovsky's. For myelin-free nerve fibers and endings, the Gross-Belynovsky, Schulze, and Rakhmanov methods are also significant. Gross-Belynovsky method (Lavrentiev modification): 1) fixation in a mixture: equal parts 96% alcohol, 1% arsenious acid and neutral formalin for an hour; 2) 10% neutral formalin—four days; 3) freezing of sections; 4) 20% Arg. nitr.—2–5 min.; 5) 4 portions of 20% formalin; 6) ammoniacal silver (microscopic control); 7) ammoniacal water (²/₃ ammonia, ²/3 water)—half an hour; 8) distilled water; 9) gold plating; 10) 5% hyposulfite, etc. - O. Schulze method: 1) formalin fixation and freezing of sections; 2) diluted distilled water normal solution of caustic soda (for fibers 10 to 50 water)—24 hours; 3) distilled water—2 hours; 4) 10% Arg. nitr.—12–24 hours; 5) reduction: 2.5 hydroquinone, 100 cm³ distilled water and 5 cm³ formalin diluted in 20, 180 and 120 times (make tests). - A. V. Rakhmanov method: 1) fixation with 96% alcohol; then absolute alcohol and paraffin; 2) sections on a slide kept at 37° for 24 hours in 10% Arg. nitr.; 3) distilled water; 4) reduction: hydroquinone 0.25, crystalline sodium sulfite 2.0, potassium carbonate 1.5, distilled water 25.0. For microglia (mesoglia)—Gortega method: 1) fixation: ammonium bromide 10.0, neutral formalin 70.0, distilled water 400.0; 2) freezing of sections; 3) distilled water with ammonia (50-2 drops); 4) ammoniacal silver: to 5 cm³ of 10% Arg. nitr. add 20 cm³ of 5% sodium carbonate; dissolve the precipitate with strong ammonia, add 15 cm³ of distilled water; 5) reduction in formalin—1 to 9 distilled water. Methods for argyrophilic fibers—Achucarro (Achucarro) and Snesarev P. E. Achucarro method: 1) fixation with 10% formalin; 2) freezing of sections; 3) distilled water; 4) saturated solution of Ac. tannici lev. puriss. in a thermostat for 15 min at 50°; 5) distilled water; 6) Belynovsky ammoniacal silver (8 drops to 20 cm³ distilled water); 7) distilled water; 8) reduction: 10% formalin. For celloid sections—Clarfeld modification. - Snesarev method—see Snesarev methods. - For calcareous deposits—Koss method: 1-- 5% Arg. nitr. 30–60 min in daylight (silver phosphate is obtained with subsequent reduction of the metal). For microbiological purposes—Levaditi and Janey methods (see Levaditi method). - It must be noted that the question of argyrophilicity of fibers, cellular granulations, etc., cannot yet be considered fully elucidated. The successes of impregnation depend extremely not only on tissues but also on the application of the indicated methods. In particular, it is very risky histologically to differentiate or identify cells by their relation to silver staining (Hamper 1).