Use of Semithin Sections Embedded in a Water-Miscible Methacrylate. for Light Microscopy of Central Nerve Tissues

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1 Okajimas Folia Anat. Jpn., 66 (2-3): , August, 1989 Use of Semithin Sections Embedded in a Water-Miscible Methacrylate for Light Microscopy of Central Nerve Tissues By Yasukazu NAGATO, Masaki SEKIGUCHI, Tsuyuka KUSHIDA, Hiroshi KUSHIDA, Kazuyo SHIMAI Department of Anatomy, School of Medicine Tokai University Department of Anatomy, Tokyo Women's Medical College -Received for Publication, March 2, Key words: Water-miscible methacrylate embedding: Hydroxypropyl methacrylate: Semithin sections: Nerve tissue: Light microscopy Summary: Semithin sections embedded in water-miscible methacrylates were used for the study of fine structures of cells and tissues in the central nervous system by light microscopy instead of the conventional paraffin sections. This method used a water-miscible methacrylate mixture consisting of 2-hydroxypropyl methacrylate (HPMA), Quetol 523 and methyl methacrylate (MMA) as an embedding medium. The mixture had a low viscosity, was easy to handle and penetrated readily and completely into the specimen, producing a homogeneous block from which it was easy than in paraffin sections owing to the thickness of the semithin sections and to the excellent structural preservation of cellular components. Shrinkage and distortion during paraffin embedding are major disadvantages in the study of structures of central nervous tissues. Morphological information from the nucleus and cytoplasmic components after paraffin sectioning was limited although various nerve cells are located in complicated brain on spinal cord tissue. To overcome these difficulties, water-miscible methacrylates were used as an embedding medium instead of the conventional paraffin embedding. The advantage of such plastic embedding is the great detail revealed as a consequence of low distortion and fine resolution not attainable with paraffin blocks. A watermiscible methacrylate, glycol methacrylate (CMA), was first developed as an embedding medium for light microscopy. Several embedding methods using GMA have been described for light microscopy of soft and hard tissues (Ruddell, 1961; Cpole and Sykes, 1974; Kushida et al., 1974, 1981; Bennett et al., 1976; Myhre and Depaoli, 1985; Hott and Marie, 1987; Liu, 1987). Ruddell (1967) first used GMA for biological specimens. Cole and Sylces (1974) embedded animal tissues in GMA using ultraviolet polymerization. Later, Kushida et al. (1975) improved the embedding method by the introduction of Quetol 523 (methoxy polyethylene glycol 400 methacrylate) and QCU-1 (2,2'-azobis isobutyronitrik paste). Quetol 523 is a light-colored, flexible, Address: Yasukazu Nagato, Department of Anatomy, School of Medicine Tokai University, Isehara, Kanagawa , Japan 145

2 146 Y. Nagato et al. water-miscible methacrylate. It has a low viscosity; it reacts with GMA and becomes an integral part of the polymerized system (Kushida et al., 1975). This embedding method allowed application of enzyme and immunohistochemical techniques (Nagato et al., 1979, 1980, 1984; Suzuki and Nagato, 1980). Adaptation for electron microscopy was also possible being semithin sections pm thick according to the method of Kushida (1977) and Kushida et al. (1981). In addition to GMA, another water-miscible methacrylate, 2-hydroxypropyl methacrylate (HPMA), has been used in limited amounts as an embedding medium for light microscopy. Recently, a mixture of HPMA, Quetol 523 and methyl methacrylate (MMA) was used as an embedding medium for observation of identical sites in semithin sections with both light and electron microscopy (Kushida et al., 1985). This method was devised for easier embedding, infiltration, sectioning and staining than GMA-Quetol 523 (Kushida et al., 1975) and GMA-Quetol 523-MMA embedding (Kushida et al., 1981) using HPMA instead of GMA. This paper describes a useful staining and related procedures using a HPMA-Quetol 523-MMA (HQM) mixture as an embedding medium for light microscopy of nerve tissue. Materials and Methods 1. Fixation. The anesthetized animals are perfused transcardially with one of the following mixture of formaldehyde and glutaraldehyde. a) 407o formaldehyde and 2% glutaraldehyde mixture b) 4% formaldehyde and 1070 glutaraldehyde mixture c) 4% formaldehyde solution (or 10% formalin) After perfusion the cerebellum was removed from the skull, cut into small blocks and immersed in the same fixative for 2-3 hr at room temperature. 2, Embedding. After fixation, the samples were washed twice with buffer, 15 minutes each, then brought to 100 7o ethanol by the graded series 50, 70, 90, 95, and ethanol, minutes each. From 100% ethanol the materials were transferred to a mixture of 100% ethanol and the resin mixture (see below) for 1-2 hr, and passed through two changes of the fresh resin mixture (see below) for 2-4 hr at room temperature using a shaker (Kushida, 1969). The embedding mixture was prepared from the following components: HPMA, Quetol 523 (Nissin EM Co., Japan and Ted Pella Inc., U.S.A.) and MMA with QCU-1 (Nissin EM Co., Japan and Ted Pella Inc., U.S.A.) as a catalyst. Quetol 523 is used as a plasticizer and reacted with HPMA. The following mixture is recommended for general embedding: 2-hydroxypropyl methacrylate 65 ml Quetol 523 methyl methacrylate 10 ml 25 ml QCU g Each of the samples was embedded in the mixture using gelatin capsules (size 00 or 0). Polymerization was then achieved in about 12 hr in an oven at 60 C. 3. Sectioning. After removal of the gelatin capsules, the block face was carefully trimmed with a clean, sharp razor blade. The trimmed blocks were cut with glass knives on a JB-4 microtome or a conventional rotary microtome. Sections were generally cut to 0.5 to 3.0 microns in thickness. In all cases dry cutting was employed. Each section was fitted from the knife edge with a pair of fine-pointed watchmaker's forceps and placed on a distilled water surface. They spread immediately to their maximum size without wrinkling. Floating individual sections were adhered to a clean slide glass without adhesive. After 10 minutes at 60 C, the dried sections were ready

3 Water-miscible methacrylate embedding of central nerve tissues 147 for staining. 4. Staining. Staining used on paraffin sections of nerve tissues can be applied successfully to HQM sections with some modification. These include hematoxylin and eosin, toluidine blue or cresyl violet for Nissl bodies, Kluver and Barrera's luxol fast blue stain for myelin and Nissl bodies, and the Feulgen reaction for DNA in nuclei. a) Hematoxylin and Eosin. Gill's hematoxylin (Polyscience, INC.) was generally used. Eosin yellowish or bluish was a 0.1% solution in distilled water. Any sections were immersed directly in the hematoxylin solution and treated according to the following procedure: hematoxylin (Gill's) 3 min rinsing with distilled water eosin 5-10 min rinsing with distilled water and drying b) Toluidine blue or Cresyl violet Toluidine blue or cresyl violet was a 0.107o solution. toluidine blue (cresyl violet) 1 min rinsing with distilled water and drying c) Kluver-Barrera's stain A 0.1% luxol fast blue solution in 95% ethanol and 0.1% cresyl violet solution in distilled water were used. luxol fast blue MBS (60 C) 1-2 hr differentiation with lithium carbonate solution and ethanol rinsing with distilled water cresyl violet 5 min rinsing with distilled water and air drying d) Feulgen reaction Hydrolysis with 5N HC1 was performed at room temperature. 5N HC1 60 min Schiff's solution 15 min washing solution 2 min each times) rinsing with distilled water and drying When completely dry, the sections were covered with a cover glass and mounted with Diatex. Results The fixative for routine use was a mixture of formaldehyde and glutaraldehyde. Although all of the fixatives employed gave good coloration with acid and basic dyes, combined 4% formaldehyde and 2% glutaraldehyde fixation (Nagato et al., 1980) showed excellent preservation of the cytological detail of each nerve cells. Toluidine blue for Nissl bodies stained the Nissl substances of Purkinje cells (Figs. 1 & 2). Hematoxylin and eosin were brilliant stains which can be used in differentiate nerve cell types and structures such as Purkinje cells, granular cells and cerebellar glomerulus (Fig. 3). Kluver and Barrera's stain was applied satisfactorily to these sections, displaying Nissl bodies, nucleoli and myelin to good advantage (Figs. 4 & 5). The Feulgen reaction was tested with all of the fixatives. Although preservation of nuclear structure was the best with a mixture of 4% formaldehyde and 2% glutaraldehyde, a mixture of 4% formaldehyde and 1070 glutaraldehyde was recommended as a fixative to prevent background staining of tissue structures. It clearly showed the structures of nucleoli or chromatin in various kind of cells (Fig. 6). Discussion A mixture of HPMA as an embedding medium was first introduced for electron microscopy (Leduc and Hott, 1965; Pease, 1966; Kushida, 1970). A mixture of HPMA, 2-butoxyethanol and triethyleneglycol (3

4 148 Y. Nagato et al. dimethacrylate was used as an embedding medium for the staining of undecalcified bone (Franklin and Hartin, 1980). Addition of triethyleneglycol dimethacrylate as a crosslinker and 2-butoxyethanol as a plasticizer resulted in relatively weak staining in the sections. Adaptation for light and electron microscopy was recently achieved by the development of a soft HPMA medium using Quetol 523 and MMA as a plasticizer (Kushida et al. 1985). Quetol 523 and MMA reacted with HPMA and became an integral part of the polymerized system without a cross-linker. In fact, a hydrophobic compound, MMA, prevented disturbance of the sections collected on the water surface. Low distortion and high stainability were also characteristic of previous GMA-Quetol 523-MMA (Kushida et al., 1981) and HPMA-Quetol 523-MMA embedded sections, but the latter had several advantages over the former. Firstly, sections embedded in the HQM mixture exhibited a slightly hydrophobic nature, i.e., they spread slowly and evenly when sections were placed on a water surface. Sections were easily removed from the knife edge or forceps without clinging or folding. This hydrophobic nature of the embedding medium made handling of the sections more convenient than that of GMA embedded sections. Secondly, components of the mixture reacted with each other and formed a uniform matrix without addition of cross-linking agents. This prevented distortion, shrinkage and swelling of the tissue sections. So destruction of nerve cells and fibers could be avoided. High magnification of a Purkinje cell, therefore, shows sharp and clear image. Absence of cross-linking agents also resulted in the flat surface of such sections. Thirdly, the viscosity of HPMA was lower than that of GMA. This physical property was beneficial for penetration of specimens. Finally, sections permitted penetration of staining dyes and reacted with tissue components due to the hydrophillic nature of the embedding matrix. However, excess staining of the embedding medium could be avoided. Sections showed no more cracking or wrinkling artifacts. In these experiments, thickness of the sections was important for visualization of the cellular structures in nerve cells. Thinner sections clearly showed cytoplasmic and nuclear detail but had less stainability. On the other hand, sharpness of the image was lost when thicker sections were used. Moreover, overlapping of tissue structure should be avoided to obtain clear images from semithin sections. Little overlapping of tissue structure also needs the preservation of cytoplasmic components more sufficiently than paraffm embedded tissue sections. Addition of glutaraldehyde improved preservation of the cytoplasmic detail when formaldehyde alone was used. Consequently, combined 4% formaldehyde and 2% glutaraldehyde fixation showed the best results in the experiments, and sections, approximately 1.5 pm thick were suitable for observing structures of nerve tissues by light microscopy. As a consequence of low distortion, tissue preservation was excellent and cellular detail was clearly observed without destruction of nerve fibers. In conclusion, the use of HQM as an embedding medium provides a useful alternative for histological studies of central nerve tissues. The another major advantage of using sections of HQM embedded nervous tissues was that the stain could be localized with low distortion and destruction of cell body and nerve fibers. Sections, 1.5 a m in thick, were satisfactory to precision in dye localization and resolution of stained sites. In particular, histological staining and identification of chromatin structures with the Feulgen reaction may be useful for classification of nerve cell types.

5 Water-miscible methacrylate embedding of central nerve tissues 149 References 1) Bennett, H.S., Wyrick, A.D., Lee, S.W., McNeil, J.H.: Science and art in preparing tissues embedded in plastic for light microscopy with special reference to glycol methacrylate, glass knives and simple stains. Stain Technol., 51: 71-97, ) Cole, M.B. Jr., Sykes, S.M.: Glycol methacrylate in light microscopy. A routine method for embedding and sectioning animal tissues. Stain Technol., 49: , ) Franklin, R.M., Martin, M.T.: Staining and histochemistry of undecalcified bone embedded in a water-miscible plastic. Stain Technol., 55: , ) Hott, M., Marie, P.J.: Glycol methacrylate as an embedding medium for bone. Stain Technol., 62: 51-57, ) Kushida, H.: A new rotary shaker for fixation, dehydration and embedding. J. Electron Microsc., 18: 137, ) Kushida, H.: A new method for embedding with 2-Hydroxypropyl methacrylate. J. Electron Microsc., 19: , ) Kushida, H.: A new method for embedding with GMA and quetol 523 for electron microscopic observations on semi-thin sections for light microscopy. J. Electron Microsc., 26: , ) Kushida, H., Kushida, T., lijima, H.: An improved method for both light and electron microscopy of identical sites in semi-thin sections under 200 kv transmission electron microscope. J. Electron Microsc., 34: , ) Kushida, T., Nagato, Y., Kushida, H.: A new embedding method employing water-miscible methacrylate for light microscopy. 10th. Int. Cong. Anat. Tokyo: 503, ) Kushida, T., Nagato, Y., Kushida, H.: New method of embedding with GMA, Queto1523 and methyl methacrylate for light and electron microscopic observation of semi-thin section. Okajimas Folia Anat. Jpn., 58: 55-68, ) Leduc, E.H., Holt, S.J.: Hydroxypropyi methacrylate, a new water-miscible embedding medium for electron microscopy. J. Cell Biol., 26: , ) Liu Chung-Ching: A simplified technique for low temperature methyl methacrylate embedding. Stain Technol., 62: , ) Myhre, J.L., Depaoli, A.: A glycol methacrylate method for the routine histologic evaluation of rat inner ear. Stain Technol., 60: 63-68, ) Nagato, Y., Kushida, T., Kushida, H.: Histochemical observation of GMA-Quetol 523 embedded tissue by light microscopy. Okajimas Folia Anat. Jpn., 56: 23-34, ) Nagato, Y., Suzuki, T., Kushida, T., Kushida, H.: Histochemical localization of alkaline phosphatase activity using GMA-Quetol 523 embedding by photopolymerization. Okajimas Folia Anat. Jpn. 57: 29-44, ) Nagato, Y., Kushida, T., Kushida, H.: Fixation of semithin section for combined light and electron microscopy. Okajimas Folia Anat. Jpn., 58: 69-80, ) Nagato, Y., Kushida, T., Kushida, H.: A method for histochemical localization of glycosaminoglycans in semi-thin tissue sections embedded in GMA, Quetol 523 and methyl methacrylate. J. Electron Microsc., 33: , ) Pease, D.C.: The preservation of unfixed cytological detail by dehydration with "inert" agents. J. Ultrastruct. Res., 14: , ) Puddell, C.L.: Hydroxyethyl methacrylatecombined with polyethylene glycol 400 and water; an embedding medium for routine 1-2 micron sectioning. Stain Technol., Q: , ) Suzuki, T., Nagato, Y.: Immunohistochemical investigation of chick oviduct using a GMA- Quetol 523 embedding method. Acta hitochem. cytochem., 13: , 1980.

6 150 Y. Nagato et al. Explanation of Figures Plate I Figs HQM embedded semithin sections of mouse cerebellum. Individual cells of cerebellar cortex are clearly seen. At a higher magnification, more cytological detail is seen to be available compared to paraffin sections. Scale in each figure is 10 micron. Fig. 1. Tissue fixed with a mixture of 4% formaldehyde and 2% glutaraldehyde. A 1.5 micron section stained with toluidine blue. Identical site in Fig. 2 is enlarged. Nucleolus and Nissl bodies of Purkinje cell are seen. Fig. 2. Same section in Fig. 1 at a lower magnification. Fig. 3. Tissue fixed with a mixture of 4% formaldehyde and 207o glutaraldehyde. 1.5 micron section stained with hematoxylin and eosin. Dendrites of Perkinje cells are clearly seen in molecular layer (arrows). Fig. 4. Tissue fixed with a mixture of 407o formaldehyde and 107o glutaraldehyde. 1.5 micron section stained with Kluver and Barrera's stain. Fig. 5. Deep granular layer and white matter of same section in Fig. 4. This section shows the detail of myelinated fibers. Fig. 6. Tissue fixed with a mixture of 4% formaldehyde and 1 lo glutaraldehyde. 1.5 micron section stained with the Feulgen reaction. The structures of nucleoli and chromatin are shown.

7 Water-miscible methacrylate embedding of central nerve tissues 151 Plate 1