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Handling Golgi-impregnated tissue for light microscopy
Neuro~ience Letters, 38 (1983) 203-208 Elsevier Scientific Pubfishers Ireland Ltd.
203
HANDLING GOLGI-IMPREGNATED TISSUI~. FOR LIGHT MICROSCOPY
PERE J. BERBEL I and ALFONSO FAIRI~N2
~Departamento de Biofisica and 2Unidad de Neuroanatomia, Instituto Cajai, CSIC, Veldzquez 144, Madrid 6 (Spain) {Received April 13th, 1983; Revised version received May Ist, 1983; Accepted May 2nd, 1983)
Key words: Golgi technique - section embedding - cortical interneurons
The use of cyanocrylic glue to fix pieces of Golgi-stained nervous tissue on a paraffin blank is proposed for obtaining thick sections of unet~tbedded tissue with a sliding microtome. This procedure makes correct orientation of the tissue easy during sectioning and makes it possible to obtain tissue sections quickly. The sections are flat-mounted u:~ing epoxy resin, resulting in permanent preparations with excellent optical properties and enabling furtt~er thin-sectioning for light and electron microscopic studies.
Current methods for sectioning Golgi-stained tissue blocks are based either on the use of celloidin or nitrocellulose embedding, or ~tn the encasing of unembedded ~|ssue in paraffin [15, 161. Additionally, a Vibratome or tissue chopper [7, 10] can be used when tissue is intended for electron microscopy. All of these methods presenf distinct advantages, but also some inconveniences. For tissue processed by most Golgi methods, embedding may result in some impairment of the Golgi impregnation, and the tissue chopper or Vibratomes may not be used for blocks presenting a large cutting face. We have found the procedure of encasing in paraffin to be more satisfactory; it is a method, used by Cajal himself [16], which can also be applied to material which is to be further processed for electron microscopy. This procedure makes correct orientation of the blocks difficult and does not allow for complete sectioning, since the last part of the block frequently becomes detached. In addition, adverse effects due to the high temperature may be expected. We describe a procedure which overcomes many of these difficulties. This procedure has been tested, so far, for the rapid-Goigi [16] and the Golgi-Kopsch [12] methods, including some variants of the latter, such as the Rio-Hortega [5], the Braitenberg [21 and the Colonnier [4] procedures. Additionally, we describe an adaptation of the section-embedding technique [3, 7, 10, 14, 17-19] which has proved to be quite efficient for bulk processing of Golgi-impregnated tissue for light microscopy, and which is also ~.pplicable to Golgi material processed for electron microscopy. After completion of the Golgi impregnation, the tissue blocks are gradually 0304-3940/83/$ 03.00 ~c: 1983 Elsevier Scientific Publishers Ireland Ltd.
204
rs m
Fig. I..%hc~rnatic dra~ing showing the Golgi-impregtmted tissue block (t) glued (arrow) to a paraffin blank (pb! attached to a rigid support trs). Usually. the support and it+; paraffin blank are kept in the holder of the microtom¢ during sectioning o f successive tissue blocks.
transferred from the silver nitrate solution to anhydrous glycerol 17]..%ctioning is by sliding microtome using a procedure in which the blocks do not need to be enc&,;ed in paraffin, resulting in better orientation. A paraffin blank is made by attaching a paraffin cube (melting point 42-46°C) to a rigid prism or support which tits in the block-holder of the microtom¢. The paraffin cube is trimmed to a truncated pyramid and several 50 #m-thick ~ctions are taken until an ¢~'en surface is obtained. This surf;~ce defines the ~ctioning plane during the ~ubsequent operations. When the paraffin blank with its support is ready, and without removing them from the microtome, the block of Golgi-impregnated tissue is blotted dry with absorbent paper and glued in the desired position to the top surface of the paraffin, using a small amount of cyanocrylic glue (Fig. I). Care should be taken to avoid an excess of glue setting around the edges of the block, which may make sectioning difficult. The glue sets in a few seconds, and the block can be sectioned. In the case of large blocks, or those containing heterogenous structures, it is advisable to give further support by placing a few drops of melted paraffin on the side opposite to that first hit by the knife. Fig. 2. Non-pyramidal neuron of layer II-III of the anterior ~.ingulate cortex of a 24-day-old mouse. Golgi-Colonnier impregnation. Like .,~me of the neurons sho~'n by Iwahori and Mizuno I I I ] it has an axon v+ith a predominantly hori,,ontal distribution. A and B: low power photomicro~aphs of the neuron in tx~,v serial, 150 ore--thick, coronal seclions. C: proximal dendrites cut by the microtom¢ knife. End points are labeled I to 4. To follow these dendrites in the section shown in B, the preparation has been rex erse-mounted to obtain a sharp focus of the cut ends of the dendrites, as shown in D; a mirror image is produced. This procedure is particularly useful to trace a.xonal branches through serial thick sections. In C, arroxv points at t.~e axon initial segment. Bars: A and B. 100 ~m; C and D, 50 ~m.
205
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~ ' ~
~
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C6' ,~
ill
•
!
•
. ~~ ,
1/11
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206
Thereafter, sections 100-150 tma thick are obtained in the usual way. The block and knife are kept moistened with glycerol and the sections are transferred, in serial order, to a petri dish containing anhydrous glycerol, where they can be stored for d
Golgi impregnation is greater and the sections mnst be mounted quickly. When the block has been sectioned, several sections of the paraffin blank are taken and, thus, the surface of the paraffin is ready to receive a new tissue block. if the Golgi sections are not intended for electron microscopy, they are transferred to a solution of glycerol and absolute ethanol (I: I) and then embedded in epoxy resin through an absolute ethanol-acetone sequence. To facilitate handling of a large number of sections, the use of an epoxy resin of relative low viscosity, such as an Epon-Araldite mixture [7J, is advised. The serial order of the sections may be maintained by individual transfer of the sections through petri dishes or aluminum weighing dishes, or by adapting the procedures described by Millhouse ItSI. Once embedded in epoxy resin, the Golgi sections are fiat-mounted [7, 10, 17, 18]. We have been using a procedure that, with some modification~ [6-8], can also be employed for electron microscopy. Briefly, the sections are placed on coverslips made of pieces of heat-resistant transparent plastic sheets with a small amount of unpolymerized resin, and carefully inverted on glass slides with a Fmmvar membrane. This membrane is made by immersing the slides in a 2¢e Formvar solution in chloroform. The epoxy resin is uniformly extended and the excess driven out while gently pushing on the coverslips with a histological needle, takinig care to maintain the serial order of sections. After polymerization, the plastic coverslip is easily removed by gently inserting the sharp point of a scalpel beneath it; the thin layer of embedding resin, containing the sections, remains adhered to the glass slide. The resulting preparations can be studied by oil-i~nmersion, from both the obverse and reverse sides [9]. When the collaterals of an axonal arborization are deep in the section and cannot be resolved approprimely, or when ne,-ronal processes must be followed through serial sections (Fig. 2), reverse observation may be desirable. The sheet of polymerized resin comaining the sections is easily removed from the glass slide using a razor blade; then the sections are reverse-mounted with a drop of water on a glass slide and dried. After observation, traces of immersion oil can be removed from ~he surface by using a miM detergent and tap water. The preparations are also suitable for using in a w icroscope with a universal stage [!], which permits the tilting of the preparation and facilitates the three-dimensional t two-dimensional projection drawings. Finally, be obtained, the sections may be attached to plastic otanKs usmg cyanocryltc glue or epoxy resin [7, 13]. Flat'mounting of Golgi-stained sections with epoxy resins overcomes many of the
207
problems associated with the traditional procedures. O f these, fading of the impregnation is the most troublesome, and to avoid this it i.s advisable to use neutral mounting media without coverslips [I 5, 16]. Indeed, preparations made in this way by Cajal himself still show a rather adequate degree of preservation. However, the tical properties, prerticularly whm the sections do not lie flat o n the slide, In addition, the use of oil-immersion objectives causes problems and frequently xylene must be used before observation to thin the layer of mounting medium over the tissue. These manipulations may gradually deteriorate the preparation. Also, synthetic mounting media (i.e. Eukitt) permit the use of coverslips, avoiding fading of the impregnation, but they have the inconvenience that resectioning of the tissue is not possible and, therefore, relationships between neurons, which only can be determined by observing semi- or ultrathin sections of Golgi-stained thick sections, cannot be elucidated [6, 8]. The procedure we describe, by its simplicity, can be applied routinely when a large number of Golgi-stained blocks must be processed to survey the neuronal organization of a given brain region by both light or electron microscopy. This work was partially supported by grants of the Fondo de lnvestigaciones Sanitarias de la Seguridad Social, and Comisi6n Asesora de lnvestigaci6n Cientifica y T~cnica. During completion of this paper, A.F. was on leave at the Laboratoire de Neuromorphoiogie, INSERM I.]-106, CMC. FOCH, Suresnes, France. Thanks to D. Le Cren for photographt,, assistance, and to Dr. J. De Felipe for discussion. t Ikrbel, P.J.. Villanueva, J.J.. Regidor, J. and Lbpez-Garcia. C., A method for the study of the spatial distribution of the neural dendritic tree using a universal stage, J. Neurosci. Meth., 4 (1981) 141+1~2. 2 Braitenberg, V., Guglielmotti, V. and Sada, E., Correlation of crystal growth with the staining of the axons by the Golgi procedure, Stain Technol., 42 (1967) 277-21!3. 3 Chang, J.P., A new technique for separation of coverglass substrate from epoxy-embedded specimens for electron microscopy, J. Ultrastruct. Res., 37 (1971) 370~377. 4 Colonnier, M., The tangential organization of the visual cortex, J. Anat. (Lond.), 98 (1964) 327-344. 5 Del Rio-Hortega0 P., Tercera aportaci6n al conocimiento morfolbgico e interpretaci6n funcional de la oligodendroglia, Mere. Soc. Esp. Hist. Nat., 14 (1928) 1-122. 6 De Felipe, J. and Fair~n, A., A type of basket cell in superficial layers of the cat t'isual cortex. A Golgi-electron microscope study, Brain Res., ??A4(1982) 9-16. 7 Fair,n, A.. Peters, A. and Saldanha. J., A new procedure for examining Golgi-impregnated neurons by light and electron microscopy. J. Neurocytol., 6 (1977) 311-337. 8 Fair, n, A. and Valverde, F., A specialized type of neuron in the visual cortex of cat: A Golgi and electron microscope study of chandelier cells, J. comp. Neurol., 194 (1980) 761-779. 9 Glaser. E.M. and Van der Loos, H., Analysis, of thick brain sections by obverse-reverse computer microscopy: application of a new, high clarity Golgi-Nissl stain, J. Neurosci. Meth., 4 (1981) 117-125. 10 Hollander, H., Target preparation for histochemical studies with the electron microscope, Science Tools, 28 (1981) 8-10.
208
st i ~ N. aad Mimmo. m.. A Go~i stud~ o,'d~ n m r o ~ oqmizmim of ~ immrtmisp4m~ corm in the mouse. II. ~ ~xm'ons, Ararat. F.mb~ol., 161 (1~1} 483-498. t2 ~ F.. F.a'atmmsm ~ r dic V ~ des F ~ d s bei der C k r o m s i l b ~ - h u ~ . Anat. Am~. II (11196)727-729. 13 I ~ m . D.A. and T~lmll. D., A procedure for smmlgi~ specific ~ of idmdfied nemom f~r ~ u ~ ~ . ~oxi. Lm.. 31 (Ira2) I-6. 14 Light, A.Ig. and I P ~ , M., T e c h t ~ , for ¢ m n l ~ lillht aml dectron microscqW,.Jf sinl~, mtragdlulal~" s t ~ tlcugons, J. Ne.dgost-i. Meth., 2 (1980) 429-430. I~ Millhouse, O.E., Th¢ Gollgi methods. In L. Hcimmr and M.J. IgoBard~ (Eds.), Nem'oanalomical Tract-Tracing Methods, Plenum Press, New York, 1981, pp. 311-J44. 16 Rangm y Cajal, S. and De Castro, F., Ekmentos de T(x-nica MicrolgrJf'ga dd Sistenta Wervimo. Salval Editmes. Barcelona. 1972. 2BJ pp. 17 Romanovicz. D.K. and Hanker. J.S.. Wafer m ~ : specimen selection in electron microscopic C)lochcmistQ- ~ith mmiophilic poly'mers. Histochcm. J.. 9 (1977) 317-327. 18 Wilson, C.J. and Grm es. P.M.. A simple and rapid section embedding technique for sequential light and electron microscopic examination of individually stained central neurons. J. Neurosci. Moth.. I (1979) JSJ- Jgl. 19 Yu,,a. A.. A transparent nonadhering silicone resin dish for embcddinlg tissues in epoxy and polyester resins. Stai;J T~hnol.. 42 (1907) J2-34.
203
HANDLING GOLGI-IMPREGNATED TISSUI~. FOR LIGHT MICROSCOPY
PERE J. BERBEL I and ALFONSO FAIRI~N2
~Departamento de Biofisica and 2Unidad de Neuroanatomia, Instituto Cajai, CSIC, Veldzquez 144, Madrid 6 (Spain) {Received April 13th, 1983; Revised version received May Ist, 1983; Accepted May 2nd, 1983)
Key words: Golgi technique - section embedding - cortical interneurons
The use of cyanocrylic glue to fix pieces of Golgi-stained nervous tissue on a paraffin blank is proposed for obtaining thick sections of unet~tbedded tissue with a sliding microtome. This procedure makes correct orientation of the tissue easy during sectioning and makes it possible to obtain tissue sections quickly. The sections are flat-mounted u:~ing epoxy resin, resulting in permanent preparations with excellent optical properties and enabling furtt~er thin-sectioning for light and electron microscopic studies.
Current methods for sectioning Golgi-stained tissue blocks are based either on the use of celloidin or nitrocellulose embedding, or ~tn the encasing of unembedded ~|ssue in paraffin [15, 161. Additionally, a Vibratome or tissue chopper [7, 10] can be used when tissue is intended for electron microscopy. All of these methods presenf distinct advantages, but also some inconveniences. For tissue processed by most Golgi methods, embedding may result in some impairment of the Golgi impregnation, and the tissue chopper or Vibratomes may not be used for blocks presenting a large cutting face. We have found the procedure of encasing in paraffin to be more satisfactory; it is a method, used by Cajal himself [16], which can also be applied to material which is to be further processed for electron microscopy. This procedure makes correct orientation of the blocks difficult and does not allow for complete sectioning, since the last part of the block frequently becomes detached. In addition, adverse effects due to the high temperature may be expected. We describe a procedure which overcomes many of these difficulties. This procedure has been tested, so far, for the rapid-Goigi [16] and the Golgi-Kopsch [12] methods, including some variants of the latter, such as the Rio-Hortega [5], the Braitenberg [21 and the Colonnier [4] procedures. Additionally, we describe an adaptation of the section-embedding technique [3, 7, 10, 14, 17-19] which has proved to be quite efficient for bulk processing of Golgi-impregnated tissue for light microscopy, and which is also ~.pplicable to Golgi material processed for electron microscopy. After completion of the Golgi impregnation, the tissue blocks are gradually 0304-3940/83/$ 03.00 ~c: 1983 Elsevier Scientific Publishers Ireland Ltd.
204
rs m
Fig. I..%hc~rnatic dra~ing showing the Golgi-impregtmted tissue block (t) glued (arrow) to a paraffin blank (pb! attached to a rigid support trs). Usually. the support and it+; paraffin blank are kept in the holder of the microtom¢ during sectioning o f successive tissue blocks.
transferred from the silver nitrate solution to anhydrous glycerol 17]..%ctioning is by sliding microtome using a procedure in which the blocks do not need to be enc&,;ed in paraffin, resulting in better orientation. A paraffin blank is made by attaching a paraffin cube (melting point 42-46°C) to a rigid prism or support which tits in the block-holder of the microtom¢. The paraffin cube is trimmed to a truncated pyramid and several 50 #m-thick ~ctions are taken until an ¢~'en surface is obtained. This surf;~ce defines the ~ctioning plane during the ~ubsequent operations. When the paraffin blank with its support is ready, and without removing them from the microtome, the block of Golgi-impregnated tissue is blotted dry with absorbent paper and glued in the desired position to the top surface of the paraffin, using a small amount of cyanocrylic glue (Fig. I). Care should be taken to avoid an excess of glue setting around the edges of the block, which may make sectioning difficult. The glue sets in a few seconds, and the block can be sectioned. In the case of large blocks, or those containing heterogenous structures, it is advisable to give further support by placing a few drops of melted paraffin on the side opposite to that first hit by the knife. Fig. 2. Non-pyramidal neuron of layer II-III of the anterior ~.ingulate cortex of a 24-day-old mouse. Golgi-Colonnier impregnation. Like .,~me of the neurons sho~'n by Iwahori and Mizuno I I I ] it has an axon v+ith a predominantly hori,,ontal distribution. A and B: low power photomicro~aphs of the neuron in tx~,v serial, 150 ore--thick, coronal seclions. C: proximal dendrites cut by the microtom¢ knife. End points are labeled I to 4. To follow these dendrites in the section shown in B, the preparation has been rex erse-mounted to obtain a sharp focus of the cut ends of the dendrites, as shown in D; a mirror image is produced. This procedure is particularly useful to trace a.xonal branches through serial thick sections. In C, arroxv points at t.~e axon initial segment. Bars: A and B. 100 ~m; C and D, 50 ~m.
205
•
t
~ ' ~
~
t
C6' ,~
ill
•
!
•
. ~~ ,
1/11
//
~,B,. ' ~
j,
206
Thereafter, sections 100-150 tma thick are obtained in the usual way. The block and knife are kept moistened with glycerol and the sections are transferred, in serial order, to a petri dish containing anhydrous glycerol, where they can be stored for d
Golgi impregnation is greater and the sections mnst be mounted quickly. When the block has been sectioned, several sections of the paraffin blank are taken and, thus, the surface of the paraffin is ready to receive a new tissue block. if the Golgi sections are not intended for electron microscopy, they are transferred to a solution of glycerol and absolute ethanol (I: I) and then embedded in epoxy resin through an absolute ethanol-acetone sequence. To facilitate handling of a large number of sections, the use of an epoxy resin of relative low viscosity, such as an Epon-Araldite mixture [7J, is advised. The serial order of the sections may be maintained by individual transfer of the sections through petri dishes or aluminum weighing dishes, or by adapting the procedures described by Millhouse ItSI. Once embedded in epoxy resin, the Golgi sections are fiat-mounted [7, 10, 17, 18]. We have been using a procedure that, with some modification~ [6-8], can also be employed for electron microscopy. Briefly, the sections are placed on coverslips made of pieces of heat-resistant transparent plastic sheets with a small amount of unpolymerized resin, and carefully inverted on glass slides with a Fmmvar membrane. This membrane is made by immersing the slides in a 2¢e Formvar solution in chloroform. The epoxy resin is uniformly extended and the excess driven out while gently pushing on the coverslips with a histological needle, takinig care to maintain the serial order of sections. After polymerization, the plastic coverslip is easily removed by gently inserting the sharp point of a scalpel beneath it; the thin layer of embedding resin, containing the sections, remains adhered to the glass slide. The resulting preparations can be studied by oil-i~nmersion, from both the obverse and reverse sides [9]. When the collaterals of an axonal arborization are deep in the section and cannot be resolved approprimely, or when ne,-ronal processes must be followed through serial sections (Fig. 2), reverse observation may be desirable. The sheet of polymerized resin comaining the sections is easily removed from the glass slide using a razor blade; then the sections are reverse-mounted with a drop of water on a glass slide and dried. After observation, traces of immersion oil can be removed from ~he surface by using a miM detergent and tap water. The preparations are also suitable for using in a w icroscope with a universal stage [!], which permits the tilting of the preparation and facilitates the three-dimensional t two-dimensional projection drawings. Finally, be obtained, the sections may be attached to plastic otanKs usmg cyanocryltc glue or epoxy resin [7, 13]. Flat'mounting of Golgi-stained sections with epoxy resins overcomes many of the
207
problems associated with the traditional procedures. O f these, fading of the impregnation is the most troublesome, and to avoid this it i.s advisable to use neutral mounting media without coverslips [I 5, 16]. Indeed, preparations made in this way by Cajal himself still show a rather adequate degree of preservation. However, the tical properties, prerticularly whm the sections do not lie flat o n the slide, In addition, the use of oil-immersion objectives causes problems and frequently xylene must be used before observation to thin the layer of mounting medium over the tissue. These manipulations may gradually deteriorate the preparation. Also, synthetic mounting media (i.e. Eukitt) permit the use of coverslips, avoiding fading of the impregnation, but they have the inconvenience that resectioning of the tissue is not possible and, therefore, relationships between neurons, which only can be determined by observing semi- or ultrathin sections of Golgi-stained thick sections, cannot be elucidated [6, 8]. The procedure we describe, by its simplicity, can be applied routinely when a large number of Golgi-stained blocks must be processed to survey the neuronal organization of a given brain region by both light or electron microscopy. This work was partially supported by grants of the Fondo de lnvestigaciones Sanitarias de la Seguridad Social, and Comisi6n Asesora de lnvestigaci6n Cientifica y T~cnica. During completion of this paper, A.F. was on leave at the Laboratoire de Neuromorphoiogie, INSERM I.]-106, CMC. FOCH, Suresnes, France. Thanks to D. Le Cren for photographt,, assistance, and to Dr. J. De Felipe for discussion. t Ikrbel, P.J.. Villanueva, J.J.. Regidor, J. and Lbpez-Garcia. C., A method for the study of the spatial distribution of the neural dendritic tree using a universal stage, J. Neurosci. Meth., 4 (1981) 141+1~2. 2 Braitenberg, V., Guglielmotti, V. and Sada, E., Correlation of crystal growth with the staining of the axons by the Golgi procedure, Stain Technol., 42 (1967) 277-21!3. 3 Chang, J.P., A new technique for separation of coverglass substrate from epoxy-embedded specimens for electron microscopy, J. Ultrastruct. Res., 37 (1971) 370~377. 4 Colonnier, M., The tangential organization of the visual cortex, J. Anat. (Lond.), 98 (1964) 327-344. 5 Del Rio-Hortega0 P., Tercera aportaci6n al conocimiento morfolbgico e interpretaci6n funcional de la oligodendroglia, Mere. Soc. Esp. Hist. Nat., 14 (1928) 1-122. 6 De Felipe, J. and Fair~n, A., A type of basket cell in superficial layers of the cat t'isual cortex. A Golgi-electron microscope study, Brain Res., ??A4(1982) 9-16. 7 Fair,n, A.. Peters, A. and Saldanha. J., A new procedure for examining Golgi-impregnated neurons by light and electron microscopy. J. Neurocytol., 6 (1977) 311-337. 8 Fair, n, A. and Valverde, F., A specialized type of neuron in the visual cortex of cat: A Golgi and electron microscope study of chandelier cells, J. comp. Neurol., 194 (1980) 761-779. 9 Glaser. E.M. and Van der Loos, H., Analysis, of thick brain sections by obverse-reverse computer microscopy: application of a new, high clarity Golgi-Nissl stain, J. Neurosci. Meth., 4 (1981) 117-125. 10 Hollander, H., Target preparation for histochemical studies with the electron microscope, Science Tools, 28 (1981) 8-10.
208
st i ~ N. aad Mimmo. m.. A Go~i stud~ o,'d~ n m r o ~ oqmizmim of ~ immrtmisp4m~ corm in the mouse. II. ~ ~xm'ons, Ararat. F.mb~ol., 161 (1~1} 483-498. t2 ~ F.. F.a'atmmsm ~ r dic V ~ des F ~ d s bei der C k r o m s i l b ~ - h u ~ . Anat. Am~. II (11196)727-729. 13 I ~ m . D.A. and T~lmll. D., A procedure for smmlgi~ specific ~ of idmdfied nemom f~r ~ u ~ ~ . ~oxi. Lm.. 31 (Ira2) I-6. 14 Light, A.Ig. and I P ~ , M., T e c h t ~ , for ¢ m n l ~ lillht aml dectron microscqW,.Jf sinl~, mtragdlulal~" s t ~ tlcugons, J. Ne.dgost-i. Meth., 2 (1980) 429-430. I~ Millhouse, O.E., Th¢ Gollgi methods. In L. Hcimmr and M.J. IgoBard~ (Eds.), Nem'oanalomical Tract-Tracing Methods, Plenum Press, New York, 1981, pp. 311-J44. 16 Rangm y Cajal, S. and De Castro, F., Ekmentos de T(x-nica MicrolgrJf'ga dd Sistenta Wervimo. Salval Editmes. Barcelona. 1972. 2BJ pp. 17 Romanovicz. D.K. and Hanker. J.S.. Wafer m ~ : specimen selection in electron microscopic C)lochcmistQ- ~ith mmiophilic poly'mers. Histochcm. J.. 9 (1977) 317-327. 18 Wilson, C.J. and Grm es. P.M.. A simple and rapid section embedding technique for sequential light and electron microscopic examination of individually stained central neurons. J. Neurosci. Moth.. I (1979) JSJ- Jgl. 19 Yu,,a. A.. A transparent nonadhering silicone resin dish for embcddinlg tissues in epoxy and polyester resins. Stai;J T~hnol.. 42 (1907) J2-34.