Chapter 1 Cytological Staining Methods R.W. Banks ■ Introduction General Introduction Neurohistology is perhaps traditionally thought of as supplying information only about the spatial or structural aspects of the neuron; however, it is my intention in the present chapter and its companion (Chapter 15 in Sect.II) to present as far as possible a unified view of neurohistology as a set of related problems centred on the relationship between structure and function of nerve cells. Such problems are not unique to the subject, but in the context of neurohistology they are uniquely complex. Neurons, as cells, are unri- valled in diversity of type, and other kinds of cells rarely match any neuron in the com- plexity of their spatio-temporal properties or in the range of genes expressed. The status of neurohistology as a recognisable discipline is therefore dependent on these proper- ties of nerve cells and nervous tissue, and its history is largely one of the development of methods aimed at overcoming the difficulties presented by them. Of course, recog- nisable disciplines need not necessarily have sharp boundaries and it is perhaps already apparent that I intend to take a fairly relaxed view as to what constitutes neurohistology. The essential criteria are whether the investigation involves the nervous system and whether it uses microscopy. Beyond those, it is a matter of taste where macroscopically neuroanatomy and neuroimaging give way to neurohistology, and microscopically neu- rohistology gives way to cellular and molecular biology. Within the discipline, boundaries must be arbitrary and harder to defend. The divi- sion into topics that can be described as cytological (this chapter) and histological sensu stricto (Chap. 15) creates such a boundary that is more convenient than real; many of the techniques covered will be applicable in either area. In a similarly cavalier fashion, I shall gather several specific techniques under rather broad and by no means exclusive headings so as to emphasize common purposes of the often disparate methods. It might be argued that the overall purpose is to provide as close as possible a description of neu- rons and nervous systems in their living state. Clearly neurohistology alone is incapable of reaching that end, but it is essential to its attainment. What is certain is that good neu- rohistology requires more than the mechanical application of various technical proce- dures aimed at a static description of the microscopic appearance of the nervous system. I suggest that what is indeed essential is the intelligent and informed combination of structural and functional elements, or at least of the interpretation of structure in func- tional terms. I hope to demonstrate the truth of this by placing several techniques in the context of specific problems in neuroscience. Any protocols and practical advice given in my chapters will be contained in these case-studies. Equally, if not more, important will be the intervening sections in which the evolutionary development and theoretical R. W. Banks, University of Durham, Department of Biological Sciences, South Road, Durham, DH1 3LE, UK (phone: +44-191-374-3354; fax: +44-191-374-2417; e-mail: r.w.banks@durham.ac.uk) 2 R.W. Banks backgrounds of various methods are briefly considered in order to highlight their pos- sibilities and limitations. The Beginnings of Neurohistology “Often, and not without pleasure, I have observed the structure of the nerves to be com- posed of very slender vessels of an indescribable fineness, running lengthwise to form the nerve” (Leeuwenhoek, 1717). Leeuwenhoek's account of his observations on the spinal nerves of cows and sheep, almost certainly the earliest histological description of a part of the vertebrate nervous system, already carries an implicit functional interpretation, for there can be little doubt that his use of the term 'vessels' is a reference to the hydraulic model of neural function proposed by Descartes (1662). His observations are all the more remarkable in view of the necessary limitation of his microtechnique to dissection with fine needles, freehand sections made with a “little knife … so sharp that it could be used for shaving”, and probably air-drying for mechanical stabilisation of tissue. Similar methods remained in virtually exclusive use for the next hundred years or so until Purkinje, who was, signif- icantly, professor of physiology at Wroclaw (Breslau), started hardening tissue in alco- hol (spirits of wine), cutting sections with his home-made microtome and staining them with various coloring agents including indigo, tincture of iodine and chrome salts (Phillips, 1987). These improvements enabled Purkinje to anticipate by two years Schwann's exten- sion of the cell theory to animals by describing nucleated “corpuscles” from a variety of tissues including brain and spinal cord (Hodgson, 1990). But new techniques rarely dis- place older ones entirely, and it was a combination of serial sectioning and microdissec- tion with needles (teasing) of chromic-acid- or potassium-dichromate-fixed tissue that allowed Deiters (1865) to demonstrate what had eluded Purkinje: the extension of the nerve cell body in dendrites (“protoplasmic processes”) of progressively finer divisions, and the continuity of the single axon with the cell body also. The problem of how to study the contextual relationships of nerve cells and their processes in situ was soon to be spectacularly solved by Golgi (1873) with “la reazione nera”, in which the use of silver nitrate was inspired, no doubt, by contemporary exper- iments in photography. Cajal took those contextual relationships to their classical limits in his magisterial exploitation of Golgi's technique (Cajal, 1995). He espoused Waldey- er's (1891) neuron doctrine in a modified and essentially modern form centred on his concept of the dynamic polarization of the neuron (Cajal, 1906). Yet his insistence on the separate identity of individual neurons had to await half a century and the development of a new technology, electron microscopy, for its confirmation (Palade and Palay, 1954). Part 1: General Histological and Cytological Methods ■■ Introduction “The principles of biological microtechnique may perhaps be reduced to one – the prin- ciple that when we make a microscopical preparation of any sort, we ought to try to un- derstand what we are doing…” (Baker, 1958). The physico-chemical, as well as the spatio-temporal, properties of living nervous tissue are not amenable to much histological work so it is generally necessary to modify Subprotocol 1 Fixation, Sectioning and Embedding 1 Cytological Staining Methods 3 them in various ways in order to produce a usable specimen. In this section we shall look at some preparative techniques that are basic to much histological study and that may be conveniently grouped under the heading of fixation, sectioning and embedding. Since they are not specific to neurohistology, the treatment of these techniques will be brief. It is particularly instructive, however, to consider them in the context of their his- torical development, which, together with that of the various methods of dyeing and staining, is typically a continuing story of progressive problem-solving by eclectic use of technologies derived from contemporary advances in other fields, principally chem- istry and physics. The natural products ethanol, in the form of spirits of wine, and acetic acid, in the form of vinegar, have always been used in the preservation of organic material, but only the first was commonly used in early microtechnique. This is because what was sought was hardening of the tissue, enabling it to be cut into thin sections, and of the two agents only ethanol had the desired effect (Baker, 1958). Hardening by the purely physical method of freezing was also possible and was used by Stilling in 1842 (cited by Cajal, 1995) to prepare sections of brain and spinal cord. With the development of inorganic chemistry in the late 18th and early 19th centuries several substances were found to harden animal tissues sufficiently to allow them to be sectioned, and their particular ef- fects were exploited either as single hardening agents or in various mixtures, many of which continue in use to the present day. The most important are – mercuric chloride, –osmium tetroxide, – chromium trioxide and – potassium dichromate, all of which were in use in microtechnique by about 1860. The subsequent rise of organic chemistry led to the introduction of the remaining classical 'hardening agents' – picric acid (2,4,6-trinitrophenol) and – formaldehyde (methanal), the latter as late as 1893 and only after its previous use as a disinfectant (Baker, 1958). As infiltration and embedding of tissue in solid media became standard practice (see below), the hardening property of these substances lost its relevance and attention could then centre on their role in fixation of the non-aqueous components of the cell. A cell that has been killed or rendered non-viable by chemical action is necessarily artefactual to a greater or lesser extent when compared to the living cell. The amount of artefactual distortion of some feature of interest in the living state can be taken as a measure of the quality of fixation in that respect, whether it be fine structure, enzyme activity, lipid ex- traction, or whatever. Moreover, in view of the physico-chemical complexity of the cell, it is not surprising that any single substance combines both good and poor fixative qual- ities when assessed on different criteria. To some extent the deficiencies of one fixative can be counteracted by the complementary benefits of another when used in combina- tion, either sequentially or together. This is necessarily an empirical process, the results of which are in general unpredictable, but it is an approach that has led to the introduc- tion of many important fixatives and fixation procedures. As an example, we shall follow the development of one of the most widely used pro- cedures, involving a combination of aldehydes with osmium tetroxide, the version in current use in Durham being given in example 2 below. Although osmium tetroxide rapidly destroys enzyme action, Strangeways and Canti (1927) found that it very faith- fully preserves the fine structure of the cell as revealed by dark-ground microscopy. Fine-structure preservation is critically important for most electron microscopy be- cause of the very high spatial resolution that it provides, so in the first two or three dec- ades of electron microscopy osmium tetroxide was widely used as the only fixative, typ- 4 R.W. Banks ically as a 1 % solution in 0.1 M phosphate or cacodylate buffer at about pH 7.3 (Glauert, 1975). It had the additional advantage of imparting electron density to those compo- nents of the specimen that reacted with the osmium tetroxide, and thus increasing im- age contrast. But the consequent loss of cytochemical information, especially about the localisation of enzyme activity which was preserved by formalin fixation (Holt and Hicks, 1961), prompted Sabatini, Bensch and Barrnett (1963) to assess various alde- hydes for their ability to preserve cellular fine structure better than formalin while re- taining high levels of enzymic action. Of the nine aldehydes assessed, including formal- dehyde and acrolein, the best results were obtained with glutaraldehyde (pentane 1,5- dial, C 5 H 8 O 2 ), which was used as a 4–6.5 % solution in 0.1M phosphate or cacodylate buffer at pH 7.2. Its superior performance is usually attributed to its relatively small size, enabling rapid penetration, and its two aldehyde groups, which are thought to allow glu- taraldehyde to form stable cross-linkages between various molecules, especially pro- teins. Moreover, when combined with a second fixation with osmium tetroxide, fine structural preservation was as good as with osmium tetroxide alone even if the blocks had been stored 'for several months' before the second step. In an early modification of the procedure Karnovsky (1965) advocated the inclusion of 4 % formaldehyde in the primary fixative, on the basis that formaldehyde, being much smaller than glutaralde- hyde and with only a single aldehyde group, would penetrate tissue more rapidly, stabi- lizing it sufficiently long for glutaraldehyde to act and thus permit the fixation of larger blocks. Whether or not this is a correct explanation for the action of the aldehyde mix- ture, the fixative has become probably the most widely used for electron microscopy, though the strength is usually reduced by half, apparently prompted by considerations of the osmotic potential of the fresh solution. Ever since Leeuwenhoek wielded his “little knife” the importance of sectioning in mi- crotechnique has been clear and, as we have seen, fixation, whether chemical or physi- cal, was initially developed to harden tissue sufficiently for it to be sectioned. Sectioning is necessary not only to make specimens suitably transparent to photons or electrons, but also to reduce the spatial complexity of a specimen to convenient limits. Analysis may be greatly facilitated, and frequently is only made possible at all, by selecting sec- tion thickness and orientation appropriate to the scale of spatial structure required of the specimen. The 3-dimensional structure of components larger than the section thickness can then be recovered by reconstruction from serial sections. But in neurohis- tology, until the discovery of the Golgi method, the complex shapes of complete nerve cells could not easily be traced in sections, and microdissection with needles of the fixed material remained in widespread use throughout much of the latter half of the 19th cen- tury. Perhaps because it is incompatible with microdissection, embedding tissue in a medium that could itself be hardened to give mechanical support during sectioning ap- pears to have been adopted relatively late into neurohistology. Embedding, when first used, was just that; the tissue was scarcely, if at all, infiltrated by the medium, but merely surrounded by it in order to retain the relative positions of separate components. Large, gel-forming molecules such as collodion (nitro-cellulose) and gelatine have been used since the earliest days of embedding when, it is no coincidence, both of these substances were also being used in the production of the first photographic emulsions. A low vis- cosity form of nitro-cellulose (“celloidin”) eventually became widely used in neurohis- tology, particularly when sections greater than about 20 µm in thickness were required. According to Galigher and Kozloff (1964), paraffin wax, a product of the then emergent petroleum industry, was first introduced as a purely embedding medium by Klebs in 1869 but almost immediately (1871) an infiltration method, essentially similar to that in current use, was devised by Born and Strickler. Neurohistologists do not appear to have taken up paraffin embedding immediately, but certainly by the end of the last decade of the 19th century it was being routinely used by them both for thin (2 µm) and serial sec- 1 Cytological Staining Methods 5 tions. Biological electron microscopy necessitated the use of new embedding media, op- portunely provided by the plastics industry from the 1940s onwards. Glauert (1975) gives a very full account of them: the most widely used are the epoxy resins. Although glutaraldehyde fixation and resin embedding were developed to meet the needs of elec- tron microscopy, the quality of their histological product is such that light microscopy has also benefited, as the following case study will show. ■■ Materials Muscle spindles partially exposed by removal of overlying extrafusal muscle fibres for direct observation in the tenuissimus muscle of the anaesthetized cat. ■■ Procedure Fixation 1. 5% glutaraldehyde in 0.1M sodium cacodylate buffer pH 7.2 for 5 min. in situ. [Glu- taraldehyde is usually obtained as a 25% solution. It polymerizes easily and so should be kept below 4 ° C until required.] 2. The same fixative for 4–14 days after excision of portions of muscle each about 10 mm long containing one spindle. [Variation in total fixation time was due to postal despatch between laboratories. There was no obvious difference in the quality of fix- ation of muscles fixed for different times.] 3. Washed in the buffer for 30 min. 4. 1 % osmium tetroxide, buffered, for 4 hours. [Osmium tetroxide penetrates tissue very slowly, but the tenuissimus muscle is typically less than 1 mm thick and could be adequately fixed in this time. OsO 4 is made up as a 2 % stock solution and kept refrigerated in a sealed bottle. The working strength fixative is made by diluting the stock solution with an equal quantity of 0.2M sodium cacodylate buffer.] Dehydration and Embedding 1. Dehydrated in a graded series of ethanol – 70 %, 95 %, 100 % (twice) – for 10 min each at ambient temperature. 2. 50:50 mixture of ethanol and propylene oxide (1,2-epoxy propane) for 15 min. [Pro- pylene oxide is usually included as an intermediate solvent and is analogous to the use of “clearing agents” in paraffin embedding procedures. The refractive index of most clearing agents is similar to that of dehydrated proteins and other cellular com- ponents; they were originally used to make fixed tissue transparent, hence the name which has persisted even though they rarely have that function today. For alternative dehydration methods see Glauert (1975).] 3. Propylene oxide for 15 min. 4. 50:50 mixture of propylene oxide and Epon (complete except for the accelerator) left overnight in an unstoppered container in a fume cupboard. [Evaporation of the pro- pylene oxide results in a very well infiltrated block.] Example 1: The Primary Ending of the Mammalian Muscle Spindle – A Case Study of the Use of 1 µ m Thick Serial Sections in Light Microscopy 6 R.W. Banks 5. Drained excess infiltration medium blotted; transfered to fresh complete Epon. 6. Flat-embedded in an aluminium foil mould; polymerized for 12 hr at 45 ° C and 24 hr at 60 ° C. Sectioning and Staining 1. Sections cut manually at 1 µ m thickness in groups of 10 on an ultramicrotome with conventional glass knives. [If necessary, the sections can be spread on the water sur- face using chloroform vapor from a brush held close to them, or by radiant heat from an electrically heated filament. Glass knives need to be replaced regularly; use of a mechanical knife-breaker ensures close similarity of shape in successive knives. Ac- curate positioning to within a few µ m of a new knife with respect to the block face can be achieved by lighting the back of the knife, such that the gap between knife edge and block face appears as a bright line.] 2. Coverslips [50x22 mm is a convenient size] scored with a diamond marker and bro- ken into strips about 3 mm wide were used to collect the sections directly from the water trough of the knife by immersing one end of the strip under the surface of the water (Fig. 1.A). [The sections, either as a ribbon or individually, are easily guided with a toothpick-mounted eyelash onto the strip, which is held in watchmakers’ for- ceps. A simple technique to ensure adequate adhesion of the sections is to draw one face of the strip of coverslip over the tip of the tongue and allow it to dry.] 3. The back of each strip was dried with a soft tissue, leaving the sections free-floating on a small drop of water on the front of the strip. 4. The sections were thoroughly dried onto the strip using a hot plate at about 70 ° C. [Best done by keeping a glass slide permanently on the hot plate and placing the strips onto the slide (Fig. 1.B).] 5. Stained with toluidine blue (Fig. 2.A) and pyronine (Fig. 2.B) at high pH by placing a drop of the stain on the sections and heating until the stain starts to dry at the edge Fig. 1. Stages in the preparation, staining and mounting of serial, 1 µm thick, epoxy resin-embed- ded sections. A: A sort ribbon of sections is guided onto a strip of glass cut from a coverslip, using an eyelash mounted on a toothpick. The strip of coverslip is held in watchmakers’ forceps. B: The back of the coverslip is dried using a soft tissue, leaving the ribbon of sections free-floating on a drop of water on the front of the coverslip, which is then placed on a glass slide on a hot-plate to flatten the sections and dry them. The same arrangement is used to stain the sections as described in the text. C: Several strips are mounted under a single large coverslip and the slide is labelled to indicate the order of the sections. 1 Cytological Staining Methods 7 of the drop. Washed with water and differentiated with 95 % ethanol. [Staining solu- tion is made by dissolving 0.1g toluidine blue + 0.05g pyronine + 0.1g borax (sodium tetraborate) in 60 ml distilled water, and should be filtered periodically.] 6. Dried on the hot plate and mounted using DPX (Distrene-Plasticizer-Xylene). [5 strips each with, say, 10 sections can be conveniently mounted under a single 50x22 mm coverslip (Fig. 1.C). Of course, the strips should be mounted with the sections uppermost.] ■■ Results The primary ending of a tenuissimus muscle spindle in the cat occupies about 350 µ m of the mid portion of the spindle and typically requires some 50 serial, 1 µm, longitudinal sections for its complete examination. The ending is generally considered to comprise the expanded sensory terminals of a single group Ia afferent nerve fibre, together with the system of preterminal branches, both myelinated and unmyelinated, that serve to distribute the terminals among the several intrafusal muscle fibres. There are common- ly six intrafusal fibres of three different kinds. Figure 3 shows a selection of micrographs taken with a x100 oil-immersion plan achromat objective (N.A. 1.25); structures consid- Fig. 2. Structural formulae of various dyes and chromogens mentioned in the text. A: Toluidine blue. B: Pyridine. C: Lucifer yellow. D: JPW1114. E: Calcium Green-1. F: FM1–43. G: DiA. 8 R.W. Banks erably less than 0.5 µ m in size are easily resolved. Each field of view covers a distance of a little over 100 µ m in the long axis of the spindle. The most prominent structure visible in the micrographs is the central portion of one of the intrafusal fibres, specifically the bag 1 . In the region of the primary ending, the sarcomeres of the intrafusal fibres are al- most entirely replaced by a collection of nuclei (Fig. 3C, n). Projecting from the surface of the fibre are the sensory terminals (Fig. 3F, t). These can be traced between the sec- tions as can portions of the myelinated (Fig. 3B, mpt) and unmyelinated (Fig. 3E, pt) preterminal branches. The dark structures within the terminals are mostly mitochon- dria. Several accessory, fibroblast-like cells are also visible forming a sheath around the bag 1 fibre. A contour line reconstruction of this part of the sensory ending on the bag 1 fibre, based on these and other intervening sections, is shown in Figure 3G. A 3-dimen- sional reconstruction of the complete ending was published by Banks in 1986. A similar Fig. 3. Examples of results of serial-section analysis using 1 µm epoxy resin-embedded material. A-F: Longitudinal sections taken in the primary sensory region of a mammalian muscle spindle. This spindle contained 5 intrafusal muscle fibres, part of only one of which (a bag 1 fibre) is shown. The sections are serial except that one section has been omitted between A and B, and one be- tween D and E. Scale bar = 10 µm. G: Contour reconstruction of the sensory terminals on the bag fibre shown in A-F. Scale bar = 50 µm. mpt, myelinated preterminal branch; n, nucleus; pt, unmy- elinated preterminal branch; t, sensory terminal. 1 Cytological Staining Methods 9 serial-section analysis was recently used by Banks et al. (1997) in a correlative histo- physiological study of multiple encoding sites and pacemaker interactions in the prima- ry ending. ■■ Introduction “The dimensions of the [synaptic] cleft are now known and its detection has led many, perhaps rather hastily, to consider the neuron (discontinuity) versus the reticular con- troversy (transynaptic cytoplasmic continuity) to be ended.” (Gray, 1964). The advent of the electron microscope removed the barrier to the study of so-called ultrastructure, or spatial organisation, on a finer scale than the resolution of the light microscope. It permitted not only synaptic clefts but also structures one or two orders of magnitude smaller to be made visible in sections of biological material. The effect on microtechnique was, however, more evolutionary than revolutionary except that obser- vation of living cells and tissues is scarcely possible with the electron microscope. It might be supposed that without the possibility of direct comparison with living cells the quality of ultrastructural fixation could only be assessed subjectively, but physical fixa- tion by rapid freezing is entirely feasible (see, for example, Verna, 1983), thus providing an objective standard for chemical methods. Freezing is not generally applicable mainly because of its limitation to very small thicknesses of tissue in order to prevent ice crystal formation (see, for example, Heuser et al., 1979), but it can be important or even essen- tial in some studies and, with sufficient ingenuity, can be applied to relatively inaccessi- ble structures within the brain (Van Harreveld and Fifkova, 1975). Despite the necessity for freezing in some special applications, ultrastructural neurohistology depends over- whelmingly on chemical fixation, the techniques being derived directly from practices and principles originally developed for light microscopy, as has been outlined above. In this section we will look briefly at the role that fixation played in the functional inter- pretation of synaptic structure. Of primary importance here was the fixation of lipids by OsO 4 , so preserving membrane structural integrity. This revealed not only the dis- continuity of neurons at the synaptic cleft, but the presence of characteristic round ves- icles of 30–50 nm diameter in the presynaptic terminals of synapses with chemically mediated transmission (Gray, 1964). The vesicles were, of course, immediately recog- nised as being correlated with, or structurally equivalent to, the neurotransmitter quan- ta. The dynamic nature of vesicle recycling during transmission was clearly established, among others, by Heuser and Reese (1973) who used immersion fixation of frog sarto- rius muscles, in a Karnovsky-type fixative, after various durations of nerve stimulation and post-stimulation recovery. Immersion fixation was initially used in ultrastructural studies on the CNS, but it was necessary to cut the tissue finely in order to obtain high quality results, so the spatial relationships of structures greater than about 1 mm in size were lost. Nevertheless, us- ing this technique, Gray (see 1964 review) was able to identify two major types of central synaptic structure and to recognize that they were differentially distributed on the den- drites and somata of the post-synaptic neurons. They were characterised by electron- dense material associated with the post-synaptic membranes that were of greater (type 1) or lesser (type 2) thickness and extent, and their locations led Eccles (1964) to suggest that they might correspond to excitatory and inhibitory synapses, respectively. Despite Subprotocol 2 Ultrastructure 10 R.W. Banks this and other important advances made using immersion fixation, the advantages of perfusion in maintaining high quality fixation while retaining larger scale structural re- lationships in the CNS are such that it very soon became the method of first choice (Pe- ters, 1970). At first veronal-acetate-buffered OsO 4 was used (Palay et al., 1962) and sub- sequently aldehydes, with or without subsequent treatment with OsO 4 (Karlsson and Schultz, 1965; Schultz and Karlsson, 1965; Westrum and Lund, 1966). Immediately, and virtually simultaneously, several authors described the occurrence of flattened presyn- aptic vesicles in some synapses. Uchizono (1965) was able to correlate round vesicles with Gray type 1 and flattened vesicles with Gray type 2 synapses; utilizing the known interneuronal origins and functional effects of certain synapses in the cerebellar cortex, he further concluded that the first were excitatory and the second inhibitory. The iden- tification was criticised on several grounds, not least that the flattening depended on al- dehyde fixation which, if prolonged, would induce even the normally round vesicles to flatten (Lund and Westrum, 1966; Walberg, 1966; Paula-Barbosa, 1975). However, many later observations have substantially confirmed Uchizono’s conclusion so that what is perhaps most interesting and instructive in this case is the usefulness of an incidental product of fixation, an artefact that without the functional correlation would otherwise be regarded as undesirable. ■■ Materials Cerebellar cortex of the adult rat, anaesthetised with an intraperitoneal injection of so- dium pentobarbitone. ■■ Procedure Fixation 1. Systemic perfusion with a Karnovsky fixative, made up as follows (proportions given for 100 ml) – Solution A: 2 g paraformaldehyde dissolved in 40 ml water at 60 ° C, 1N NaOH add- ed dropwise (2–6 drops) until the solution clears. – Solution B: 10 ml of 25 % glutaraldehyde mixed with 50 ml of 0.2M sodium ca- codylate buffer, pH 7.3. Solutions are kept at 4 ° C until required, then mixed to give 100 ml complete fixa- tive. Techniques of perfusion vary considerably in their elaboration; the method I have adopted is simple and seemingly reliable: it aims to minimise the time be- tween induction of anaesthesia and effective fixation. A peristaltic pump [Watson- Marlow MHRE 200] is used to provide the driving force [many authors use hydro- static pressure] and the fixative is introduced immediately the cannula is in place, beginning at a relatively low speed until signs of onset of fixation are evident (limb and tail extension), and progressively increasing the speed over the first few min- utes. Fixation is continued for about 10 minutes, consuming about 500 ml fixative for an adult rat. Pressure is not monitored. The cannula is fashioned from a 21G hypodermic needle, angled at its mid-point and ground transversely at the tip. A blob of epoxy resin applied to the tip before Example 2: Synapses of the Cerebellar Cortex [...]... describe a modified SAGE protocol (Protocol B) that requires minimal amounts of starting material, making it extremely suitable for use in neuroscience Using this protocol we can obtain an expression profile from a single hippocampal punch derived from a 300-µm brain slice, which we estimate to contain at least a factor 5x103 less polyA+ RNA than is required for the original procedure (protocol A) 29 30 Erno... very long (10 to 800 bp) compared to the classic PAA electrophoresis gels (10 to 300 bp or 150 to 500 bp) and thus less PAA-gels are required As the protocols are rather specific for this equipment and as it is delivered with an excellent and detailed protocol explaining how to use it, we will here restrict ourselves to some general remarks 1 To accurately compare cDNA fragments, load PCR samples generated... Widespread use of tissue slices and of the confocal microscope, together with continued development of the applications of reactive and other dyes, have increased the importance of fluorescent markers in neuroscience One might also cite technical advances in microelectronics and computing as necessary factors The markers have made it possible to study various aspects of the activity of living neurons,... around the dye molecules in the nerve membrane” Acridine orange, incidentally, is one of a family of dyes and pharmacologically active substances originally developed in the search for anti-malarial agents Modern “voltage-sensitive” dyes are more likely to be specifically sought; one such designed for intracellular applications is JPW1114 (Figure 2D), which has a particularly high signal:noise ratio among... P 1983 The section-Golgi impregnation procedure 1 Description of the method and its combination with histochemistry after intracellular iontophoresis or retrograde transport of horseradish peroxidase Neuroscience 9, 463–474 Galigher, A E and Kozloff, E N 1964 Essentials of Practical Microtechnique Henry Kimpton, London Glauert, A M 1975 Fixation, Dehydration and Embedding of Biological Specimens North-Holland,... Heimer, L and RoBards, M J (eds.) Neuroanatomical Tract-Tracing Methods Plenum, New York, pp 311–344 Morest, D K 1981 The Golgi methods In Heym, Ch and Forssmann, W.-G (eds.) Techniques in Neuroanatomical Research Springer, Berlin, pp 124–138 Muller, K J and McMahon, U J 1976 The shapes of sensory and motor neurons and the distribution of their synapses in ganglia of the leech: a study using intracellular... rat slices J Chemical Neuroanat 6, 311–321 Onn, S.-P., Pucak, M L and Grace, A A 1993 Lucifer yellow dye labelling of living nerve cells and subsequent staining with Lucifer yellow antiserum Neurosci Protocols 93-050-17-01-14 Palade, G E and Palay, S L 1954 Electron microscope observations of interneuronal and neuromuscular synapses Anat Rec 118, 335–336 Palay, S L., McGee-Russell, S M., Gordon, J... 1970 The fixation of central nervous system and the analysis of electron micrographs of the neuropil with special reference to the cerebral cortex In Nauta, W H J and Ebbesson, S O (eds.) Contemporary Research Methods in Neuroanatomy Springer, Berlin, pp 56–76 Phillips, C G 1987 Purkinje cells and Betz cells Physiol Bohemoslov 30, 217–223 Pinault, D 1994 Golgi-like labeling of a single neuron recorded... preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation J Cell Biol 17, 19–58 Scheibel, M E and Scheibel, A R 1978 The methods of Golgi In Robertson, R T (ed.) Neuroanatomical Research Techniques Academic, New York, pp 89–114 Schiller, J., Schiller, Y., Stuart, G and Sakmann, B 1997 Calcium action potentials restricted to distal apical dendrites of rat neocortical pyramidal... The section-Golgi impregnation procedure 2 Immunocytochemical demonstration of glutamate decarboxylase in Golgi-impregnated neurons and in their afferent synaptic boutons in the visual cortex of the cat Neuroscience 9, 475–490 Stean, J P B 1974 Some evidence of the nature of the Golgi-Cox deposit and its biochemical origin Histochemistry 40, 377–383 Stewart, W W 1981 Lucifer dyes – highly fluorescent . the truth of this by placing several techniques in the context of specific problems in neuroscience. Any protocols and practical advice given in my chapters will be contained in these case-studies (Cajal, 1995). He espoused Waldey- er's (1891) neuron doctrine in a modified and essentially modern form centred on his concept of the dynamic polarization of the neuron (Cajal, 1906). Yet. nervous tissue are not amenable to much histological work so it is generally necessary to modify Subprotocol 1 Fixation, Sectioning and Embedding 1 Cytological Staining Methods 3 them in various ways