BENJAMIN F. TRUMP, 9 EDWARD A. SMUCKLER, 2 and EARL P. BENDITT. Department of Pathology, University of Washington,. Seattle, Washington.
J. ULTRASTRUCTURE RESEARCtt: 5, 3 4 3 - 3 4 8
(1961)
343
A Method for Staining Epoxy Sections for Light Microscopy 1 BENJAMIN F. TRUMP,9 EDWARD A. SMUCKLER,2 and EARL P. BENDITT
Department of Pathology, University of Washington, Seattle, Washington Received February 17, 1961 A technique for staining sections of osmium-fixed, epoxy-embedded tissues for light microscopy is presented. The method employs aqueous toluidine blue at pH 11.1 and does not require prior removal of embedding medium. When stained with this technique and viewed with an oil immersion objective, the images are striking because of their great definition and resemblance to electron micrographs. Such a method is useful for identification and mapping of areas seen in the electron microscope; it also permits better utilization of the full resolving power of the light microscope. Severe sampling problems are encountered in electron microscopic studies, particularly those of pathological material. These can be partially alleviated if a thick section (0.5-2 #) is taken adjacent to the thin section (0.02-0.05 #) and used for identification and mapping of the area with light microscopy. For this purpose either phase contrast microscopy or staining of sections has been utilized. Several methods for staining sections of methacrylate-embedded tissue have been devised (1, 4, 6, 9, 11), most of which involve removal of the embedding resin with a suitable solvent. The problem is somewhat different with tissues embedded in epoxy resins as there are presently no solvents for these after polymerization; staining must therefore be accomplished without prior removal of embedding medium. Staining with the resin in situ has in addition the advantage of avoiding the cytologic distortions produced by solvent removal of the embedding medium. Richardson et al. (8) recently presented a method of staining epoxy resin-embedded material with periodic acid and Mallory's Azure II methylene blue. We wish to present another method for staining epoxy resin-embedded tissue sections for light microscopy which produces patterns of relative densities which have a striking resemblance to low-power electron micrographs. The advantage of this method is the greater ease of matching corresponding areas in the light and electron microscopes and a simpler staining procedure. This work was supported in part by a grant-in-aid from U.S.P.H.S. (H-3174). Post-doctoral Trainee in Experimental Pathology. 2 3 - 61173319 J . Ultrastruct~tre l~e~earch
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BENJAMINF. TRUMP, EDWARD A. SMUCKLER, AND EARL P. BENDITT
Small cubes of tissue are fixed for 2 hours at 0°C in 2 % OsO4, buffered with scollidine (2) to a p H of 7.4. The tissues are d e h y d r a t e d with a g r a d e d series of alcohols a n d e m b e d d e d in E p o n e p o x y resin a c c o r d i n g to the technique of Luft (7). Sections 0.5-2 # thick are cut with glass knives using a P o r t e r - B l u m microtome; they are transferred to a d r o p of water on a l b u m i n i z e d glass slides using a fine brush, a wire loop, or a s h a r p e n e d a p p l i c a t o r stick. The slides are then d r i e d at r o o m t e m p e r a t u r e for 24 hours o r more. The sections should be stained as follows. (1) F l o o d the slide with a freshly p r e p a r e d , filtered aqueous solution of 0.1% toluidine blue in 2.5% Na2CO ~ ( p H 11.1) a n d allow to stand for 30-120 minutes. W i t h i n 15 minutes the sections are readily visible as b l u e - v i o l e t areas against a b l u e p i n k b a c k g r o u n d . W h e n the desired interval of time has elapsed, the sections are e x a m i n e d with a light microscope. If the staining is sufficiently intense the cells are d a r k reddish-purple. A t this point, however, there is m u c h b a c k g r o u n d staining a n d cytologic details are p a r t i a l l y obscured. (2) G e n t l y remove the staining solution by inclining the slide a n d wiping the excess f r o m a r o u n d the section with gauze or filter p a p e r ; the section m a y be gently blotted. (3) W a s h gently with t a p water b y flooding the slide with a medicine d r o p p e r ; incline the slide a n d r e m o v e the effluent as described above. (4) I n a similar m a n n e r wash very briefly with 90 % a l c o h o l a n d then with a b s o l u t e alcohol. B o t h washes serve to differentiate the stain a n d the section should again be e x a m i n e d u n d e r a microscope. If staining is n o t sufficiently intense, or if t o o m u c h dye has been extracted, the entire staining p r o c e d u r e m a y be repeated.
Fins. 1, 3 and 4 are photomicrographs taken from 1-2-# sections of osmium-fixed tissue embedded in Epon epoxy resin and stained with toluidine blue. Fro. 2 is an electron micrograph of an ultrathin section adjacent to that shown in Fig. 1. Fie. 1. Collecting tubule from the kidney of a normal rat. The lumen (L) is at the top. Portions of six epithelial cells are seen (Ep). The mitochondria (M), which appear as round black structures in the photograph, are dark purple. The epithelial cell nuclei (N1-N~) are lavender and have dark purple nucleoli (Nu). The tubular basement membrane (B) is lavender. Below the tubule is an interstitial cell (FI), probably a fibroblast. In the cytoplasm of this cell are many small vacuoles (arrows) which represent dilated cisternae of the endoplasmic reticulum as can be seen in the electron micrograph (Fig. 2). × 2250. FIG. 2. Electron micrograph of a thin section adjacent to that shown in Fig. 1 showing the cells outlined in the light micrograph. The lumen (L) is at the upper right. A few small, poorly-developed microvilli (V) are present along the luminal margins of the cells. Many mitochondria (M) and membrane-limited cytoplasmic droplets (D) are visible within the cytoplasm of the epithelial cells. The lateral intercellular membranes (/) are tortuous. A small portion of the nucleolus (Nu) is visible within one of the nuclei (N). The tubular basement membrane (B) separates the tubules from the interstitial tissue; within the latter is a fibroblast (F~) with an elongate nucleus. At the left, within the cytoplasm of the fibroblast, prominent dilated portions of endoplasmic reticulum can be seen (E). Note the similar distribution of densities in this electron micrograph and the light micrograph of Fig. 1. × 7400.
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BENJAMINF. TRUMP, EDWARD A. SMUCKLER, AND EARL P. BENDITT
(5) Clear rapidly with xylene and m o u n t in P e r m o u n t or balsam. A l t h o u g h the procedure m a y also be carried out in Coplin jars, the above procedure of flooding the slides with the reagents is more satisfactory since the sections less frequently float loose. Further, the use of horizontal flooding helps salvage a loose section. A n o t h e r m e t h o d of avoiding this has been tried which consists of m o u n t i n g the unstained sections on slides which have previously been coated with a thin layer of epoxy resin (3). This is accomplished by spreading a thin layer of the m o n o m e r mixture on clean glass slides, and polymerizing the resin by placing the slides in a 60 ° oven for 30 minutes. The sections are m o u n t e d by floating them onto a drop of water which is placed on the resin-coated slide, a In our hands this m e t h o d does not yield sections with the resolution of the albuminized slide process. Some wrinkling of the section often occurs. This can be reduced or eliminated if sections are air-dried between each step in the staining procedure. W h e n viewed with an oil immersion objective, the images are very striking because of their resemblance to low-power electron micrographs (Figs. 1 and 2). The distribution of densities is similar following both these procedures. Objects of relatively high density in electron micrographs, such as mitochondria (Figs. 1 and 3), nucleoli (Fig. 1), erythrocytes (Fig. 4), and cytoplasmic protein droplets (Fig. 1), are dark purple; objects of lesser density, such as nuclei (Fig. 1) and basement m e m b r a n e material (Fig. 1,B and Fig. 4,B), are lavender. Lipid droplets appear in various shades of green (Fig. 3, L 1 and L~). Metachromasia is observed in mast cell granules. The mechanism of staining is not clear; the pattern is not that which is usually associated with toluidine blue. Although the staining observed m a y be related to the action of osmium on the tissues during fixation, further studies are needed to define the reactions involved. A similar pattern of staining is obtained if buffers at lower Although various mixtures of epoxy resin would probably be satisfactory for this purpose, we have found the resin mixture marketed by Sears-Roebuck & Co. to be particularly convenient.
Fla. 3. Liver cell from a rat which had received 5.0 cc/kg body wt. of carbon tetrachloride by stomach tube 6 hours previously. The cytoplasm contains many large vacuoles (V). Electron micrographs of adjacent thin sections show that these vacuoles represent dilated cavities and cisternae of the endoplasmic reticulum (10). The mitochondria (M) which are dark black in the photograph stain purple. The two large lipid droplets (L1 and L2) in the center of the cell are bright green in the stained preparations. In the lower left is a portion of a sinusoid containing an erythrocyte (E) which is dark purple, x 2400. FI6. 4. Portion of a normal rat renal glomerulus. Four capillary lumens (L1 L~) are present, three of which contain erythrocytes (Ri-R3). Five endothelial cell nuclei (E~-Es) are present and, along the luminal aspect of the glomerular basement membrane (B), portions of attenuated endothelial cytoplasm are indicated by arrows. Cytoplasm of epithelial cells (EP) is clearly visible on the outside of the basement membrane and in several areas the outline of the foot processes (FP) are visible. Bowman's space (BS) and epithelial cells of the parietal layer of Bowman's capsule (PE) can be seen at the lower left. x 3609.
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BENJAMIN E. TRUMP, EDWARDA. SMUCKLER, AND EARL P. BENDITT
pH values are used to prepare the staining solutions. The staining is much less intense, however, and therefore less satisfactory from the standpoint of cytologic detail. In addition to its usefulness in mapping and identification of areas seen in the electron microscope, this method applied to light microscopy allows better utilization of the resolving power of the instrument than that obtainable with paraffin sections. The authors gratefully acknowledge the technical assistance of Mrs. Nancy Krall Trump and Mr. Kermuth L. Montgomery. REFERENCES 1. BENCOSME,S. A., STONE, P. S., LATTA, H. and MADDEN, S. C., J. Biophys. Biochem. Cytol. 5, 508 (1959). 2. BENNETT,H. S. and LUFT, J., 3". Biophys. Biochern. Cytol. 6, 113 (1959). 3. CHAMBERS,V., personal communication. 4, CHURG, J. and GRISHMAN,E., J. Mt. Sinai Hosp. N. Y. 24, 736 (1957). 5. Houcg:, C. E. and DEMPSEY,E. W., Stain Technol. 29, 207 (1954). 6. JENNINGS, B. M., FARQUHAR,M. G. and MOON, H. D., Am. or. Pathol. 35, 991 (1959). 7. LUET, J., J. Biophys. Biochem. Cytol. 9, 409 (1961). 8. 'RICHARDSON,K. G., JARRETT,L. and F1NKE, E. H., Stain Technol. 35, 313 (1960). 9. RUNGE, W. J., VERNIER, R. L. and HARTMANN,J. F., J. Biophys. Biochem. Cytol. 4,
327 (1958). 10. SMUCKLER,E. and ISERI, 0., Biochem. Biophys. Research Commun. (in press). 11. THOEMES,W. M., Mikroskopie 64, 406 (1960).
