A New Method for Intense Staining of Myelin

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1 Volume 46(4): , 1998 The Journal of Histochemistry & Cytochemistry TECHNICAL NOTE A New Method for Intense Staining of Myelin Karen J. McNally and Alan Peters Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston, Massachusetts SUMMARY We describe a new method for intense staining of myelin. The stain involves immersing frozen or vibratome sections in 4% normal horse serum. A DAB reaction is then carried out, which results in the deposition of reaction product in myelin sheaths. On intensification of this reaction product using the silver enhancement technique described by Görcs, myelin stains an intense black color, making the preparations suitable for photography. The stain is especially useful for determining the distribution of myelinated fibers in gray matter. (J Histochem Cytochem 46: , 1998) KEY WORDS myelin horse serum DAB reaction silver intensification This myelin staining method was discovered as we were carrying out a series of control trials on vibratome sections of central nervous system (CNS) tissue to determine the specificity of labeling produced by a variety of antibodies. We found that when sections were treated with horse serum, which is normally used to block unspecific labeling, and when the primary antibody stage was omitted from the procedure, the subsequent diaminobenzidine (DAB) reaction caused the white matter and some components in the gray matter to become a light brown. To determine the elements in the tissue in which DAB reaction product was deposited, we used the silver enhancement technique first published by Gallyas et al. (1982) and given in more detail in Görcs et al. (1986). Examination of the sections revealed that the material being labeled by the DAB reaction is myelin, which stained black after the intensification procedure had been used. Because the staining of the myelin was so intense, we evaluated the usefulness of this method as a myelin stain in sections of CNS taken from rat, cat, and monkey. The tissue had been fixed by a variety of methods. Other laboratories have also tried this technique and have been sufficiently impressed with the results to suggest that we publish the details of the procedure. A brief account of the procedure has been published previously in articles by Peters and Sethares Correspondence to: Dr. Alan Peters, Dept. of Anatomy and Neurobiology, Boston U. School of Medicine, 80 East Concord St., Boston, MA Received for publication September 4, 1997; accepted November 4, 1997 (7T4460). (1996) and Peters et al. (1997), in which the stain was used to evaluate the disposition of myelinated fibers in the visual cortex of the monkey. Materials and Methods This stain has been used both on frozen sections, after cryoprotection, and on vibratome sections taken from the brains and spinal cords of rats, cats, and monkeys. The central nervous system tissue used has been fixed by a variety of methods, including the following: (a) intracardiac perfusion with 1.25% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M phosphate or cacodylate buffer at ph 7.4. Before being sectioned, tissue from these brains was usually stored in a solution containing twice the above strengths of aldehydes; (b) immersion fixation or perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer at ph ; and (c) immersion fixation in 10% buffered formalin containing 4% (w/v) formaldehyde and M phosphate buffer (Fisher; Pittsburgh, PA) It is recommended that sections are stained while floating free in the solutions, because if they are mounted on glass slides before being stained, the stain tends to be uneven. It is also important not to treat the sections with alcohol before passing them through the staining procedure, because this destroys their ability to be stained, perhaps by extracting some components from the myelin. Staining Procedure for Light Microscopy Initial Staining Reaction. Rinse sections in 0.1 M Tris phosphate-buffered saline (TPBS) at ph 7.4 on a shaker table at room temperature twice for 30 min. Place the sections in a solution of 4% normal horse serum and 0.5% Triton X with TPBS. Sections are left overnight at room temperature on a shaker table. The Histochemical Society, Inc /98/$

2 542 McNally, Peters Figure 1 Cross-sectioned cervical spinal cord of a monkey, showing the staining of myelinated fibers. Bar 0.5 mm. Figure 2 Hippocampus of a rat in a section of the cerebral hemisphere taken in the frontal plane. To the left is the dentate gyrus (d) with a core of myelinated fibers. The gyrus is separated from the heavily stained white matter (w) of the cerebral cortex by Ammon s horn (A). Bar 0.5 mm. Figure 3 A section passing through the depth of the striate cortex of a monkey. The pia is at the top. In the cortex are vertically oriented bundles of myelinated fibers (arrows), and the horizontal band of staining halfway through the depth of the cortex is the line of Gennari (g). Bar 0.5 mm.

3 A New Myelin Stain 543 Rinse sections in TPBS twice for 15 min on the shaker at room temperature. Place sections in diaminobenzidine (DAB) (kit containing Tris buffer, DAB, and hydrogen peroxide; Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 9 min. Rinse sections in TPBS twice for 10 min. At this stage the myelin is only faintly visible. The staining is intensified with the silver intensification procedure given by Görcs et al. (1986). Intensification of the DAB Deposit. Place sections in 8% thioglycolic acid in distilled water in a fume hood on a rotator, either for 4 6 hr at room temperature or overnight at 4C. Wash sections by rotating them in 2% sodium acetate in distilled water four times for 10 min. (Leaving sections in 2% sodium acetate for longer than the recommended four times for 10 min results in a noticeable increase in the developing time.) Place sections in the developing solution (see below for composition) until the desired degree of intensity of staining is attained. We have found that the developing time is usually 8 10 min, but monitor sections carefully by light microscopy, because developing times may be longer, even among sections from the same animal. If the development is not carefully monitored the sections can be overstained. Rotate sections in 1% acetic acid (Fisher; 30-A-36) in distilled water for 5 10 min. Rotate sections in 0.05% gold chloride (Fisher; G-54) in distilled water for 15 min. Rinse sections in 2% sodium acetate (Fisher; S220-1) in distilled water for 30 sec. Rotate sections in 3% sodium thiosulfate (Fisher; S474-3) in distilled water for 5 min. Rinse sections in 0.1 M phosphate buffer, ph 7.4, twice for 10 min. If a more intense staining is required, repeat the protocol beginning at the intensification procedure. After staining has been completed, sections can be mounted either by bringing them through a graduated series of glycerin to 100% and mounting them on a glass slide, which is then coverslipped and the edges sealed with nailpolish, or by mounting the stained sections on subbed glass slides, after which they are allowed to air-dry and coverslipped using Permount. The composition of the developing solution is as follows: Solution A 5% sodium carbonate (Sigma, St Louis, MO; S-6139) in distilled water Solution B 1000 ml distilled water 2.0 g ammonium nitrate (Sigma; A-9642) 2.0 g silver nitrate (VWR, Philadelphia, PA; VW6030-1) 10.0 g tungstosilic acid (Fluka, Milwaukee, WI; Chemika 95395) Solution C 37% formaldehyde (Fisher; F77P-20) Combine Solutions A, B, and C as follows. Gradually add 40 l of Solution C to 10 ml of Solution B, stirring continuously while adding. Then add this resulting mixture of B and C dropwise to 10 ml of Solution A, stirring continuously. This final solution should be clear. If the final solution becomes cloudy, discard the developing solution and make a new one. Cloudiness will either cause a dramatic increase in the developing time or produce no reaction. Staining Procedure for Electron Microscopy Follow the procedure for light microscopic staining of vibratome sections, but use only 0.01% Triton X in the initial staining procedure with 4% normal horse serum and TPBS. Process the sections as described, but osmicate the sections in 1% osmic acid, dehydrate, and mount the sections in araldite between sheets of aclar so that they can be sectioned for electron microscopy. Results Examples of the kind of staining obtained using this technique are shown in Figures 1 4. The sections are taken from the spinal cord (Figure 1), and striate cortex (Figures 3 and 4) of a monkey and from the cerebral hemisphere of a rat (Figure 2). In stained preparations, myelin is intensely black, so that white matter is well differentiated from gray matter (Figures 1 and 2). However, individual fibers are so well stained that they are readily visible as they pass through the gray matter (Figures 2 and 3). At higher magnification it is evident that only the myelin and not the enclosed axons is stained, because in cross-sectioned fibers the myelin sheath is seen as a black ring surrounding the unstained axon (Figure 4, arrows) and in obliquely sectioned fibers the myelin sheath is evident as two parallel black lines on each side of the axon (Figure 4, arrowheads). To further ascertain the specificity of this stain, some of the stained sections from the cerebral cortex of a monkey that had been perfused with a mixture of aldehydes were examined by electron microscopy. As shown in Figure 5, the deposit of metallic particles that is produced by the intensification procedure is confined to the myelin sheaths of the nerve fibers. The fact that a metallic deposit is produced by this staining procedure for myelin means that the stain is very stable and, unlike some dyes, it is not likely to fade with time. Figure 4 A horizontal section through the striate cortex of the monkey taken at the level of layer 4C, just below the line of Gennari. The myelinated fibers in the vertical bundles of axons are shown in cross-section and in some of them the myelin appears as a ring around the unstained axon (arrows). Other fibers have been sectioned obliquely (arrowheads). Bar 25 m.

4 544 McNally, Peters Figure 5 Electron micrograph of a myelin-stained preparation from the striate cortex of a monkey showing the specificity of the stain. The myelin sheaths contain gold particles (arrows) that replace the silver that is laid down in the intensification stage of the procedure. Bar 2 m. It should be added that when this stain has been used on tissue derived from old animals, light microscopy shows that some granules stain in the neuropil. We have examined these granules by electron microscopy and found that they are produced by stain being deposited on the phagocytic inclusions within neuroglial cells. Discussion The specificity of this stain is made evident by its disposition in sections taken from a variety of different areas of the CNS, and by the fact that in electron microscopic preparations the metallic particles are present only within the myelin sheaths. It is assumed that these metallic particles are gold, because although silver is laid down in the intensification procedure it is replaced by gold when the sections are immersed in the gold chloride solution, any remaining silver subsequently being removed by the sodium thiosulfate. This reaction is similar to the gold toning reaction used by Fairén et al. (1977), in which gold is used to replace the silver chromate deposit generated by the rapid Golgi impregnation. However, as far as could be determined, the particles are not specifically related to any single component of the sheath. As pointed out above, though, in light microscopic preparations from the cerebral cortices of old monkeys over 25 years of age, the stain can also label phagocytosed material within neuroglial cells (Peters et al. 1997). Whether the stained phagocytosed material is degenerating myelin is not known, but in this context it is of interest that Gerrits et al. (1992) found that when they used Sudan Black B to stain the spinal cords of rats with chronic experimental allergic encephalomyelitis, the stain labeled both myelin sheaths and degenerated myelin contained within macrophages. It is not evident why horse serum, or a fraction of the serum, binds to components of myelin, nor is it clear why DAB should deposit at the binding sites. It might be conjectured that there is an oxidizing agent in the fraction of horse serum that binds to the myelin and its derivatives, and that this causes oxygen to be released from the hydrogen peroxide in the DAB solution to catalyze the polymerization of the DAB, so that the resulting deposit can be visualized using intensification. Other normal sera, such as normal goat serum, will also produce myelin staining, but the reaction is not so intense as with the normal horse serum. The value of the stain that we have described here is that the staining of the myelin is very intense. Even individual fibers are clearly visible, so that the stain is particularly useful for examining the distribution of nerve fibers in gray matter (Peters and Sethares 1996).

5 A New Myelin Stain 545 This stain is excellent for examining small numbers of frozen or vibratome sections. However, if sections are mounted on slides before staining, the resulting stain tends to be patchy. In addition, treating sections with alcohol and other dehydrating agents before staining destroys the stain. Consequently, this is not a stain that will replace the routine procedures, such as the many varieties of the Weigert (1884) hematoxylin stains (e.g., Lillie 1965), the more commonly used Luxol Fast Blue (Klüver and Barrera, 1953), which can be used on serial sections cut from embedded material, or the recently published method by Schmued (1990), in which frozen sections are incubated in gold chloride. In this latter method, frozen sections of formalin-fixed tissue are incubated in a gold chloride solution, and it is interesting that, as with the horse serum stain being described here, Schmued (1990) points out that soaking sections in alcohol before incubating them in gold chloride eliminates the myelin staining. In summary, we have described a new stain for myelin, based on soaking sections in normal horse serum and then visualizing the myelin with an intensified DAB reaction. The method is especially useful for staining sections in which intense staining of fibers in gray matter is required, and it is very suitable for photography. Acknowledgments Supported by Program Project Grant AG from the National Institute on Aging of NIH. We are grateful to Dr Douglas Rosene for his helpful critique of earlier versions of this article, for carrying out extensive trials on the use of this stain, and for comparing its suitability with that of other routine myelin stains. Literature Cited Fairén A, Peters A, Saldanha J (1977) A new procedure for examining Golgi impregnated neurons by light and electron microscopy. J Neurocytol 6: Gallyas F, Görcs T, Merchenthaler I (1982) High-grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry. J Histochem Cytochem 30: Gerrits PO, Brekelmans Bartels M, Mast L, s-gravenmade EJ, Horobin RW, Holstege G (1992) Staining myelin and myelin-like degradation products in the spinal cords of chronic experimental allergic encephalomyelitis (Cr-EAE) rats using Sudan Black B staining of glycol methacrylate-embedded material. J Neurosci Methods 45: Görcs TJ, Léránth C, MacLusky NJ (1986) The use of goldsubstituted silver-intensified diaminobenzidine (DAB) and nonintensified DAB for simultaneous electron microscopic immunoperoxidase labeling of tyrosine hydroxylase and glutamic acid decarboxylase immunoreactivity in the rat medial preoptic area. J Histochem Cytochem 34: Klüver H, Barrera E (1953) A method for the combined staining of cells and fibers in the nervous system. J Neuropathol Exp Neurol 12: Lillie RD (1965) Histologic Technic and Practical Histochemistry. 3rd ed. New York, McGraw Hill Peters A, Cifuentes JM, Sethares C (1997) The organization of pyramidal cells in area 18 of the rhesus monkey. Cerebral Cortex 7: Peters A, Sethares C (1996) Myelinated axons and the pyramidal cell modules in monkey primary visual cortex. J Comp Neurol 365: Schmued LC (1990) A rapid, sensitive histochemical stain for myelin in frozen brain sections. J Histochem Cytochem 38: Weigert C (1884) Ausführliche Beschreibung der in no. 2 dieser Zeitschrift erwähnten neuen Färbungsmethode für das Centralnervensystem. Fortschr Deutsch Med 2: