a detailed methodological description of the ...

A C T A U N IV E R S IT A T IS L U N D E N S I S

1965

S E C T IO II

No.

7

M E D IC A , M A T H E M A T IC A , S C IE N T IA E R E R U M N A T U R A L IU M

A D ETAILED M ETH O D O LO GICAL DESCRIPTION OF TH E FLU O R ESCEN CE METHOD FOR THE C E L L U L A R DEM O N STRATIO N OF BIO G EN IC M O NO AM IN ES BY

B E N G T F A L C K a n d C H R IS T E R O W M A N FROM THE DEPARTMENTS OF HISTOLOGY AND ANATOMY UNIVERSITY OF LUND

LU N D

1965

C.W .K . G L E E R U P , S W E D E N

Read before the Royal Physiographic Society, February

LUND 1965 HÅKAN OHLSSONS BOKTRYCKERI

jo ,

1965

Contents I n t r o d u c tio n .......................................................................... 5 Present m e t h o d .................................................................... 6 General considerations................................................... 7 Preparation of tissues.........................................................7 Freeze-drying apparatus................................................... 8 Histochemical p r o c e d u r e ............................................10 Embedding and sectioning............................................ n Fluorescence m ic ro sc o p y..................................................12 Microscopic differentiation between various mono amines ............................................................................. 12 Differentiation of specific fluorescence from auto fluorescence ...................................................................13 Summary of pro ced u re....................................................... 14 Preparation of tissues . . ............................................ 14 Freeze-drying procedure..................................................15 Histochemical and histological procedure . . . 16 For staining after fluorescence microscopy . . . 16 Stretch-preparations of thin tissu e s ........................... 17 S u m m a r y ............................................................................. 17 A ckn o w led gem en ts.............................................................17

R e fe re n c e s.................................................................................17

Contents I n t r o d u c tio n ..........................................................................5 Present m e t h o d ....................................................................6 General considerations................................................... 7 Preparation of tissues.........................................................7 Freeze-drying apparatus................................................... 8 Histochemical p r o c e d u r e ............................................io Embedding and sectioning............................................ i i Fluorescence m ic ro sc o p y..................................................12 Microscopic differentiation between various mono amines ..............................................................................12 Differentiation of specific fluorescence from autofluorescence ...................................................................13 Summary of pro ced u re....................................................... 14 Preparation of tissues....................................................... 14 Freeze-drying procedure................................................. 15 Histochemical and histological procedure . . . 16 For staining after fluorescence microscopy . . . 16 Stretch-preparations of thin tis s u e s ........................... 17 S u m m a r y ............................................................................. 17 A ckn o w led gem en ts.............................................................17

R e fe re n c e s.................................................................................17

Introduction Many basic problems concerning the biogenic monoamines D A , N A , A and 5-H T1 demand for their solution, methods which permit direct demonstration of the monoamines themselves at the cellular level. For this reason an investi gation into the histochemical possibilities of detecting the stores of neuronal monoamines was started several years ago in this laboraty in co-operation with Dr. N.-A. H illarp (Dept, of Histology, Karolinska Institutet, Stockholm) and Dr. A . C arlsson (Dept, of Pharmacology, University of Göteborg). A t first, this task did not seem very encouraging because of the very great sensitivity and specificity that must be inherent in such methods. The various procedures that have long been available are more or less unspecific, their reaction mecha nisms are often poorly understood, and all have a low sensitivity. However, despite such limitations, some of these methods have been valuable for studying chromaffin cell systems: e. g. the chromaffin reaction, the iodate method of H illarp and H ökfelt (19 55) for the specific demonstration of N A , and the fluorescence method of E rös (19 3 2 ), H am perl (19 3 2 ) and E ränkö (19 5 2) by means of which cells storing 5-HT and N A can be demonstrated providing they contain large quantities of the amines. The situation became more hopeful when it was shown to be possible to develop a highly sensitive histochemical procedure ( C arlsson et al., 19 6 1) based on the principle that certain C A :s can be con verted to highly fluorescent trihydroxyindoles. Although, because of technical difficulties, this method could not at this stage be used for detecting e. g. neuronal monoamines, it suggested the possibility of developing useful fluores cence microscopic methods. Early in 1961 the development of another method began, which within a short time showed itself to possess unusual sensitivity, specificity and applica bility for demonstrating tissue monoamines. Because of these properties, not only could the adrenergic transmitter be visualized for the first time ( F a lck and T orp, 1962) but it also became possible to localize hitherto unknown cellular stores of monoamines.

1 Abbreviations used in this paper include: A (adrenaline); C A (catecholamine); D A (dop amine); DOPA (3,4-dihydroxy-phenylalanine); 5-HT (5-hydroxytryptamine); 5-HTP (5hydroxytryptophan); N A (noradrenaline). Acta Univ. Lund. II. 1965. No. 7 .

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Bengt Falck and Christer Owman

Present method In attempts to introduce principles such as that of H ess and U denfriend (1959) for fluorimetric determinations of tryptamine [condensation with formaldehyde and subsequent oxidation to the strongly fluorescent norharmane) into histo chemistry, a very unexpected observation was made. As could be predicted, practically no fluorescence could be obtained when C A :s were treated in solu tion or on paper with aqueous or gaseous formaldehyde, but the primary amines were found to convert readily into intensely fluorescent products in formaldehyde gas under very mild conditions providing the reaction took place in the presence of dry protein. It was also found that the compounds formed, became enclosed in, and perhaps also partly bound in the protein [through methylene bridges to reactive groups) in such a w ay that they were not ex tracted by organic solvents or even by the lower alcohols [ F a lc k et al., 1962). This peculiar fluorescence reaction is the basis of the histochemical procedure [ F a lc k , 1962). The fluorescence method has been thoroughly studied, especially with the use of model systems, and its chemical and histochemical background and specificity are now well understood [ C orrodi and H illarp , 1963, 1964, C orrodi et al., 1964). The first step in the fluorescence reaction is a condensation with the formation of a 6,7-dihydroxy-i,2,3,4-tetrahydroisoquinoline. Under the mild conditions used, great activation by the 3-OH group of the side chain is required for this ring closure, and obviously the C A :s must be either primary or secondary amines. Their 3-O-methylated and acid metabolites therefore do not react. OH-groups in both 3 and 4 positions are needed to obtain products with an intense fluorescence. The next step is a quite unexpected proteincatalyzed dehydrogenation giving finally the fluorescent product, a 6,7-dihydroxy-3,4-dihydroisoquinoline, with main peaks of activation and emission at 410 and 480 mu, respectively [uncorrected instrument values; C orrodi and H illarp , 1963). In the case of a secondary amine, e. g. A , this dehydrogenation gives rise to a quaternary 3,4-dihydroisoquinoline and requires more severe reaction conditions. Primary and secondary C A :s may thus be differentiated from each other histochemically in a simple way. Not only the C A :s [and D O PA) but also 5-HT [and 5-HTP) react readily and in the same fundamental manner as described above. Only those indolic compounds with a tryptamine side chain and an unsubstituted 2-position will condense with formaldehyde, indoles with 5-hydroxy and 5-methoxy groups forming condensation products with high fluorescence intensity. The fluorescent product of 5-HT, 6-hydroxy3,4-dihydro-/?-carboline, has an emission spectrum with a peak at about 530 mu [uncorrected instrument values; C orrodi and H illarp , 1963). C A :s and 5-HT can consequently be distinguished in the fluorescence microscope by their Acta Univ. Lund. II. 1965. No. 7 .

A Detailed Methodological Description

7

different fluorescence colours (green and yellow, respectively) if certain con ditions and precautions are observed (see p. 12 ). The chemistry of the fluore scence reaction, as well as other investigations ( H am berger et al., 1964, H illarp and M almfors , 1964) disprove the view of C sillik (1964) that the method has the serious limitation that only those amines bound in the storage granules can be demonstrated. Extensive studies of model systems and tissues have shown that the method has a high specificity for C A :s and 5-HT. With the help of several histochemical criteria— based on the characteristics of the fluorescence reaction and the properties of the fluorescent products—almost conclusive evidence can usually be obtained that an observed fluorescence is due to the presence of such amines. It is also possible to differentiate between primary and secondary amines, and between C A and 5-HT. Analyses of activation and emission spectra, by means of a new microspectrophotometric technique developed by C aspersson , have proved to be of great value both for evaluation of specificity and for differentiation between C A and 5-HT ( C aspersson , H illarp and R itzen , to be published). The equipment and procedures, which were devised in this laboratory unless otherwise stated, have been in constant use, with small modifications, during the last three years, and reproducible and consistent results have been achieved by standardizing both the freeze-drying and histotechnical procedures. How ever, many investigators have encountered technical difficulties when trying to reproduce this method from directions that have been given in previous pub lications. This has prompted the present detailed technical description of the method, including practical considerations.

General considerations It is of fundamental importance that all procedures, including the histochemical reactions, are performed in a dry milieu. The low sensitivity of earlier methods for the demonstration of monoamines, e. g. the chromaffine reaction, is due inter alia to the use of fluid fixatives and reagents in solution. Moreover, the final step in the formation of the fluorophore upon treatment with formaldehyde does not even occur at all if the reaction is performed in a solvent system ( C orrodi and H illa rp , 19 63). For this reason, the preparations are rapidly brought to the temperature of liquid nitrogen and freeze-dried, and then the whole dry piece of tissue is treated in formaldehyde gas, allowing the histo chemical reaction to occur (with a simultaneous fixation of the specimen) before paraffin embedding and sectioning.

Preparation of tissues The animal is killed and the organs are dissected out as quickly as possible. No difference in the result has been observed whether the animal is killed Acta JJniv. Lund. II. 1965. No. 7 .

8


with a blow on the neck without previous anaesthesia, by decapitation during light ether anaesthesia, or by removing the organs from the living animal anaesthetized with nembutal. It may be of value to note that perfect results have also been obtained from slaughter house preparations as much as 60 min after the death of the animal. In order to avoid the formation of ice crystal artifacts during the freezing procedure, the tissue piece is frozen rapidly to a very low temperature ("quench ing” ). The growth of ice crystals ceases at temperatures below - 3 0 ° to - 5 0 0 C. If this temperature range is passed rapidly by quenching to lower temperatures, only submicroscopic, if any, ice crystals are formed. It is, however, not possible to drop the specimen directly into liquid nitrogen, because the transference of heat from the preparation is very much reduced by the formation of a layer of vaporized nitrogen on its surface. Freezing is therefore performed by the use of various intermedia ( P earse , 19 6 1, M oline and G len n er , 1964). A mixture containing propane and propylene in roughly 9: 1 proportions, kept at the temperature of liquid nitrogen (Fig. 1 A ), has been found to be convenient for the present purposes, though other proportions of propane and propylene may well be found to be equally suitable. Little or no boiling occurs when introducing the specimen into this mixture, which has a boiling point of about - 4 5 o C. A convenient source of the gas mixture is the commercially available propane, e. g. for domestic use, which is often, though not always, contaminated with an appropriate amount of propylene. Pure propane solidifies at the temperature of liquid nitrogen and is thus useless for quenching purposes. After quenching in the propane mixture, the preparation is rapidly trans ferred to the small compartments of a metal block which is kept immediately beneath the surface of liquid nitrogen in a Dewar vessel (Fig. 1 B ). The specimens may be kept in the liquid nitrogen for as long as necessary and when all the preparations have been collected, the whole metal block is rapidly trans ferred to the drying apparatus. It is possible to use cryostate sections of quenched tissues for special pur poses ( H am berger and N orberg , 1964), but this technique results in more or less pronounced diffusion of the monoamines (see illustrations in C sillik and E r u lka r , 1964) and cannot be recommended for this reason. Thin tissue sheets, such as iris, mesentery or meninges from smaller animals, can be spread across slides, dried for a short time at room temperature in air or in vacuo, and, after treatment in formaldehyde gas, analysed directly in the fluorescence microscope. For practical details of the procedures, see p. 14 and p. 17.

Freeze-drying apparatus A num ber o f good m odels o f freeze-d ryers are available (cf. P earse , 19 6 1) and n e w typ es h ave recently been developed, e. g. the therm oelectric apparatus o f Acta Utiiv. Lund. II. 1965. No. 7 .


9

P earse (196 3) and the new device of T hieme (1965) which has been extensively tested in this laboratory with excellent results. Many of these models do, of course, fulfil the present demands, but they are often expensive and generally have only a small capacity. In this presentation, only one type will be described (Fig. 2). It has been designed in the present laboratory, and has been found to give excellent drying of the preparations, it is easily handled, and it allows the simultaneous treatment of any reasonable number of specimens. Freeze-drying implies the removal of water from rapidly frozen tissues by sublimation at a temperature below the freezing-point of the tissue (cf. N eu m a n n , 19 58 ]. Vacuum is maintained around the solid tissue piece to allow vaporization of its water. In order to obtain efficient drying, including removal of the residual water, about 2-4 % ( N eu m an n , 1958), a trap must be provided for the continuous removal of the water molecules. This is achieved in the present apparatus by a "cold finger” (Fig. 4 A , b ), on the surface of which the vaporized water is condensed. The distance between the specimen and the condensing surface (35-50 mm), and the vacuum in the apparatus, are adjusted with respect to the “mean free path” (for definition, see P earse , 19 6 1). A twostage mechanical pump (Fig. 2, c) in combination with an oil-diffusion pump (Fig. 2, d) provides a vacuum of about io -6 mm Hg. This is a higher vacuum than is necessary for freeze-drying but it gives an efficient insulation around the inner tube (Fig. 3, b ). The consumption of cooling mixture is further reduced by using a silvered column of an appropriate length (Fig. 3 ). The cooling mixture is solid carbon dioxide and acetone, giving a temperature of -8 7 ° C. The drying temperature, i. e. the temperature at which the preparations are kept during the drying procedure, is maintained at a constant level by cooling an ethanol bath (Fig. 4 A , e) with a coil (Fig. 2, h) from a thermostatically regulated (Fig. 4 A , c) compression-expansion unit (Fig. 2, g). This is a more convenient and reliable arrangement than to use cooling mixture for the adjust ment of the drying temperature. V ery effective drying without any displacement of the monoamines from their cellular stores can be obtained at temperatures as high as -2 0 0 C. Different diameters may be used for the outer tube of the column and in large-sized dryers, up to 100 preparations have been dried at a time. A very useful size of dryer is shown in Fig. 3, which accomodates about 45 tissue pieces. The preparations are collected in a metal block (Fig. 1 B, c) which fits into the glass cup closing the outer tube of the dryer (Fig. 3, and 4 B , d). This metal block is manufactured by melting lead and tin in equal proportions. The molten alloy is poured into the glass cup, previously heated to + 3 0 0 ° C in an oven. Small burr holes (Fig. 1 B, c) in which the preparations are placed after quenching, are made on its upper surface. Alternatively, small metal baskets or plastic cylinders about xo mm high may be cemented on the surface of the metal block (Fig. 5, d ). A metal hook for lifting the block is screwed into the Acta Univ. Lund. II. 1965. No. 7 .

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centre (Fig. i B, d). The same block is always used with the cup in which it was made, so providing the greatest possible contact surface, and when placed in the dryer the block provides a very efficient conductor of heat between the specimens and the outer ethanol bath. Moreover, the block remains cold enough to prevent thawing of the preparations when it is transferred from the Dewar vessel to the drying apparatus. When drying is completed, the temperature around the preparations must be raised well above room-temperature to avoid condensation of water upon them; they are now, of course, very hygroscopic. Since the water has been removed, there is no risk of diffusion of substances within the specimen even after this rise in temperature, provided the preparations are kept in a dry milieu. After stopping the vacuum pumps, air is let into the system via a cold trap (Fig. 2, f) to maintain a dry milieu for the preparations. For practical details of the procedure, see p. 15.

Histochemical procedure After freeze-drying is completed, the metal block with the preparations is rapidly transferred to a closed perspex box (Fig. 5) containing dishes of phos phorus pentoxide (Fig. 5, b) to absorb the moisture. If necessary, the specimens may be kept in this dry compartment for a few days at room temperature in darkness, although they should be treated with formaldehyde gas as soon as possible. They are handled in the box from the outside via holes provided with rubber cuffs (Fig. 5, a) and are transferred to separate, numbered receptacles (Fig. 5, f) made from a perspex ring with a fine meshed bottom (e. g. of nylon net). The specimens are now fragile and should be carefully handled with fine entomologists’ forceps (Fig. 5, e). The receptacles are placed in 1 litre glass vessels containing 5 gm para formaldehyde. The vessels are sealed with plastic lids and are heated in an oven at + 8 o ° C (primary C A :s can react at lower temperatures, ca + 6 o ° C, but a temperature of + 8 o ° C is used routinely). During heating, the para formaldehyde is depolymerized to gaseous formaldehyde. Besides the specific condensation reaction with the monoamines, yielding the intensely fluorescent derivatives, the formaldehyde treatment also gives a mild but sufficient fixation of the tissues. Primary C A :s and 5-HT develop a maximal fluorescence within 1 hr, but for A it is necessary to continue the formaldehyde treatment for up to 3 hr. The remaining paraformaldehyde is discarded after use. The water content of the paraformaldehyde is of critical importance for the outcome of the histochemical reaction. Thus, paraformaldehyde batches are stored in desiccators at different but constant, relative humidities for at least 5-7 days before use. This can be ensured by using various concentrations of Acta Univ. Lund. II. 1965. No. 7 .


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sulphuric acid in the desiccators (cf. H odgm an et al., i960). The strongest reaction is obtained with paraformaldehyde that has been stored at a high relative humidity (80-95 % ) , but then the fluorescent structures are often diffuse. If the paraformaldehyde has been stored at low relative humidities (below 30 % ), the reaction, if it develops at all, is distinct, but very weak. Optimal results are usually obtained if the paraformaldehyde is stored over sulphuric acid giving a relative humidity of about 50-70 % . It should be noted that various types of tissue and even various parts of the same tissue, may behave differently towards different types of paraformaldehyde. The outcome of the reaction is also dependent upon the degree of dryness of the specimen after freeze-drying. It is therefore often advisable to test several pieces of the tissue to be studied, obtained from the same dryer, in different paraformal dehydes, and to choose the one giving the optimal reaction. The fixation obtained by treatment in formaldehyde gas instead of solution is excellent, and it minimizes diffusion and extraction of components in the tissues. Freeze-drying and formaldehyde gas treatment can therefore be recom mended for any type of histological or histochemical work where formaldehyde fixation is permissible. For example, aldehyde fuchsin has been found to give a much more brilliant staining of neurosecretion by this method than after fixation in fluid fixatives. The preparations are sensitive to humidity even after formaldehyde treat ment, and should therefore be stored in desiccators preferably in darkness, if embedding does not follow shortly afterwards. For practical details of the procedure, see p. 16.

Embedding and sectioning The specimens should embedded in degassed paraffin w ax as soon as possible after formaldehyde treatment. Embedding is performed in vacuo to ensure complete and rapid penetration of the dry tissue. A container shown in Fig. 6 is used for this purpose. The vessel consists of two chambers connected by a standard ground glass fitting, and a side arm with a stop-cock that can be con nected to a mechanical vacuum pump. One chamber (a) contains paraffin w ax and is immersed in the water bath; the specimen is placed in the second chamber (b) which is then connected to chamber a. The vessel is evacuated and the specimen tipped into the melted paraffin wax. After blocking the preparation in paraffin, it is sectioned and mounted on dry slides in Entellan (Merck) containing some extra xylene to dissolve the paraffin of the sections. When A is to be studied, the sections must be mounted in liquid paraffin since the fluorophore of A is soluble in organic solvents such as xylene ( F a lc k et al., 1963, O w man and S jô st ra n d , 1965). Mounting has a negligible immediate effect on observable fluorescense intensity and is usually Acta Univ. Lund. II. 1965. No. 7 .

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necessary to avoid light-scattering in the sections which might be misinterpreted as specific fluorescence. Sections that have not been deparaffinized may be stored in a dry place in darkness for a few weeks. Once mounted, however, they should be analysed in the fluorescence microscope as soon as possible, since a considerable decrease of the fluorescence intensity is often encountered after 24 hr. For practical details of the procedure, see p. 16.

Fluorescence microscopy The fluorophores of the C A :s and 5-HT all have the same activation peak between 390 and 410 m¡1. However, the fluorescence spectrum of the dihydroisoquinoline formed during formaldehyde treatment of the C A :s ( C orrodi and H illa rp , 1963] shows a peak at 480 m u , whereas the dihydro-/3-carboline formed from 5-HT under similar conditions has a peak 30 to 40 m/< higher. These two types ■ of fluorophores can therefore be distinguished in the fluo rescence microscope by their different colours (green and yellow, respectively),

providing a secondary filter with a high absorption below 490 m u is used in the microscope tube to exclude the blue component. Microscopic analysis can be carried out with an ordinary light microscope with suitable accessories for fluorescence microscopy (e. g. Leitz, Reichert, Carl Zeiss). A high pressure mercury lamp (Osram, HBO 200) serves as the source of UV-light. The light is passed through a heat absorption filter (Schott K G 1 ) and a Schott B G 12 filter of 3 -7 mm thickness (too thin a filter will cause a very disturbing blue background fluorescence). The light is then focused by a metallized front-surface mirror (a glass-covered mirror causes considerable absorption of the activation light and decreases fluorescence intensity) onto a dark-field condensor. An oil-immersion condensor gives much higher fluore scence intensity than a dry condenser system. The emitted light is filtered through a Schott O G 4 (1 mm thick), or equivalent filters. A much higher fluorescence intensity is observed with a monocular than a binocular tube. For further details on the general principles of fluorescence microscopy, se Y o ung (19 6 1). For photomicrography Gevaert Scientia 50B65, Scopix G or Kodak Ekta chrome EHB films are suitable.

Microscopic differentiation between various monoamines It is possible to differentiate directly in the fluorescence microscope between C A :s and 5-HT, as well as between primary and secondary C A :s. Acta Univ. Lund. II. 1965. No. 7 .


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1. The colour of the C A (and D O P A ) fluorophores is green though some times with a yellowish tinge, while those of 5-HT (and 5-HTP) are unmistakebly yellow. It should be observed, however, that cells storing very large amounts of C A :s (“chromaffin cells”) develop a green-yellow to brilliant yellow, or even a reddish fluorescence (in the case of A , these colour changes appear only after prolonged formaldehyde treatment) and then a differential diagnosis may be difficult. 2. The fluorophores of C A :s are less sensitive to exposure to UV-light from the microscope lamp than the yellow fluorophore of 5-HT which often rapidly decreases; e. g., in 5-hydroxytryptaminergic nerves this decrease is perceptible after only 1- 2 min exposure to UV-light. Certain cell systems storing large amounts of 5-HT, do not show this rapid decrease in fluo rescence. 3. If freeze-dried tissues are treated in formaldehyde gas saturated with water, the C A :s diffuse from their cellular sites before their condensation with formaldehyde and binding to the protein have time to occur. This effect is not so pronounced in the case of 5-HT (see experiments by B ertler et al., 1964). 4. Primary and secondary C A :s can be distinguished because: a) primary C A :s develop their maximal fluorescence after a much shorter treatment with formaldehyde than A does (see p. 10 ); b) the fluorophore of A is extracted more or less completely after mounting the sections in media containing organic solvents.

Differentiation of specific fluorescence from autofluorescence Many tissues contain structures which display a brown to yellow, or even an intensely green autofluorescence. Such material may have a similar cellular distribution to the specific monoamine fluorescence, and direct differentiation may sometimes be difficult. There are several ways, however, of differentiating between specific monoamine fluorescence and unspecific autofluorescence: 1. Autofluorescent material generally differs in colour from that of the mono amine fluorophores. 2. The autofluorescence is fairly stable towards UV-light, although there are some exceptions, e. g. the red autofluorescence of the Harderian gland in the orbit of the rat and mouse, which rapidly fades. 3. Autofluorescent material is present in specimens treated in heat but without formaldehyde. Autofluorescence in tissues is often greatly diminished or even suppressed by the treatment with formaldehyde gas. Acta Univ. Lund. II. 1965. No. 7 .

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4. If "w et” paraformaldehyde is used, the monoamine fluorophores are seen to have a diffuse distribution or even to disappear, while the autofluo rescence remains unaffected. 5. The autofluorescence remains unaffected by treatment with pharmacolo gical agents which decrease (e. g. reserpine) or increase (e. g. monoamine oxidase inhibitors) the monoamine stores. 6. The specificity of the fluorescence can be tested chemically by the sodium borohydride test ( C orrodi et al., 1964).

Summary of procedure Preparation of tissues 1. The propane-propylene mixture is condensed in a metal cup suspended in liquid nitrogen [big- 1 A ) . The gas is led from the gas cylinder by a copper tube which coils first in the liquid nitrogen before dipping into the cup. The appropriate length of this tube will be found by experience; if it is too long, the gas pressure in the cylinder will be insufficient to force the liquified propane mixture into the cup. Carry out the entire process in a fume cupboard and cover the container with a cloth when the operation is completed to prevent water and oxygen condensation into the propane mixture so avoiding explosion risks. 2. Suspend the metal block in a metal frame just beneath the surface of liquid nitrogen in a second Dewar flask (Fig. 1 B) and cover the container with a cloth. 3. Kill the animal and dissect out the tissue pieces. The pieces should be as small as possible; specimens as large as a mouse brain can be dried but they very often crack more or less severely when quenched. Place the specimens on small strips of paper with code numbers written on the back for identification. The paper should be glazed and not porous (i. e. not filter paper). 4. Holding the paper strip with forceps, transfer the specimen to a wire gauze basket in the cooled propane mixture. The specimen should remain well below the surface of the propane for at least xo sec. The gauze basket, with a suitable handle, simplifies retrieval of the pieces. 5. Remove the specimen from the liquid propane mixture and transfer as quickly as possible to a numbered depression in the metal block suspended in liquid nitrogen. The specimens remain in liquid nitrogen until trans ferred to the freeze-dryer. Add extra liquid nitrogen, as necessary, to keep the surface just above that of the metal block. Acta Univ. Lund. II. 1965. No. 7 .


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Freeze-drying procedure 1. Tw o or three hours before freeze-drying begins, switch on the compressor unit (Fig. 2) to reduce the temperature of ethyl alcohol (96 % ] in the Dewar flask to - 3 5 0 to -4 0 0 C. Suspend the glass cup in the alcohol bath (Fig. 4 A ) and seal with a plastic lid, using high vacuum silicone grease, to prevent water condensation on the inner surface of the cup. Place about 200 ml acetone in the cold finger (Fig. 3). 2. Remove the lid from the cooled cup and transfer the metal block con taining the preparations from liquid nitrogen to the bottom of the cup. Replace the plastic lid and wait for two or three minutes to allow residual liquid nitrogen in the depressions of the metal block to boil away. Grease the flange of the outer tube of the column with high vacuum silicone grease. 3. Start the backstage pump (Fig. 2]. Remove lid, and seal the cup to the bottom of the column; hold in position until fixed by the vacuum (Fig. 4 B ). 4. Raise the alcohol bath as soon as possible, and adjust it so that the cup is well down in the alcohol (Fig. 4 C ). Set the thermostat control of the compressor unit (Fig. 4] to -2 0 0 C ; this temperature is maintained throughout the process of drying. 5. The vacuum should become effective after about 20 min. Check the vacuum with a high frequency tester and switch on the oil diffusion pump (Fig. 2]. 6. Add crushed solid carbon dioxide to the inner tube (Fig. 3) and fill to within about xo cm of the top. 7. Check the vacuum with a high frequency tester daily, and measure the level of dry ice in the column with a ruler. It may sometimes be necessary to add more dry ice towards the end of the period of freeze-drying, but an abnormally large loss indicates leakage in the vacuum system. Freeze-drying is usually completed within 2 -5 days, depending upon the size and type of tissue. 8. On the day before breaking the vacuum, switch off the compressor. 9. On the final day, replace the alcohol bath and its cooling coil by a thermo statically controlled water bath at + 3 5 0 C for 4 hr. During the final hour, play warm air from two hair dryers onto the joint between the outer tube of the column and the cup, to reduce the viscosity of the vacuum grease in the seal and so facilitate removal of the cup. 10. Fifteen min before breaking the vacuum, switch off the oil diffusion pump and allow it to cool. Place a small Dewar flask of liquid nitrogen around the cold trap (Fig 2). 11.

Open the air inlet slowly. Remove the cup, taking care not to strike it against the cold finger, and seal with a plastic lid.

Acta Univ. Lund. II. 1965. No. 7 .

16 12.


Transfer the metal block with the preparations quickly into the dry-air box (Fig. 5).

Histochemical and histological procedure 1. Transfer the specimens from the metal block to separate, numbered com partments in plastic containers. This operation is carried out in the dry-air box over phosphorus pentoxide, via holes fitted with rubber cuffs, and the specimens are handled with fine entomologists’ forceps. 2. Transfer the plastic containers with the specimens to 1 1. glass jars con taining 5 gm paraformaldehyde. Seal the jars with plastic lids and heat in an oven at + 8 o ° C for 1 - 3 hr. 3. Store the plastic containers with the specimens in a desiccator over phos phorus pentoxide in darkness if embedding does not follow immediately. 4. Place paraffin w ax C52°_54° C m.pt] in one arm of the special container (Fig. 6) and melt in a water bath at + 6 o ° C. Place the specimen in the other arm of the container which is then connected to the first vessel. Evacuate the container, and tip the specimen into the paraffin w ax by tilting the whole vessel. 5. Infiltration is completed in 10 min. Remove the specimen with forceps and prepare block. 6. Clean slides in dichromate solution overnight. Wash in running water for 12 hr, rinse in alcohol, and dry. 7. Place the paraffin sections on a dry slide. Transfer the slide to a hot plate at + 6 0 ° C for a short time, allowing the paraffin to melt and the sections to spread. 8. Remove the slide from the hot plate and allow the paraffin to harden again. For tissues containing adrenaline: 9a. Cover the sections with liquid paraffin and add cover-slip. 10a. Return slide to the hot plate for at least 30 min. in order to dissolve the paraffin w ax into the liquid paraffin. For other tissues: 9b. Cover the sections with a few drops of Entellan-xylene mixture (2-3 ml xylene in 30 ml Entellan] and add cover-slip. 10b. Return the slide to the hot plate for 20 min. The paraffin is completely dis solved by the mounting medium.

For staining after fluorescence microscopy 1. After sectioning, spreading sections, and cooling (steps 7-8 above], dip the slide briefly in 0 .5 -1 % celloidin in ether:alcohol mixture (50: 50). Allow the slide to drain for a short time (it is easier to get too little than too much celloidin and the slide must not drain for too long]. Acta Univ. Lund. II. 1965. No. 7 .


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2. Air-dry for a short time; the celloidin film should not become white. 3. Deparaffinize in xylene and mount in liquid paraffin for fluorescence microscopy. 4. After fluorescence microscopy, remove cover-slip and if the sections appear loose, dip in celloidin again. 5. Dissolve liquid paraffin with xylene. 6. Stain and mount, following normal histological procedures. Longer staining times than usual may be necessary because of the celloidin film.

Stretch-preparations of thin tissues 1. 2. 3. 4.

Spread tissue membranes on clean microscope slides. D ry in desiccator over phosphorus pentoxide for 1 hr. Treat in formaldehyde gas from optimal paraformaldehyde batch. The tissue can be mounted in Entellan or liquid paraffin for fluorescence microscopy, but this is not essential.

Summary A methodological description gives detailed instructions for the preparation, freeze-drying, histochemical treatment, and sectioning of tissues for fluorescence microscopy of catecholamines, 5-hydroxytryptamine and their immediate pre cursors.

Acknowledgements The development of the procedures was made possible by grants from Air Force Office of Scientific Research under Grant A F EO A R 64-5 through the European Office of Aerospace Research (O A R ) United States Air Force; United States Public Health Service (NB 05236 -01); Association for the Aid of Crippled Children, New York, U .S.A .; and the Swedish Medical Research Council.

References B ertler, Å., F alck, B.,

and O wman , C h .: Studies on 5-hydroxytryptamine stores in pineal gland of rat. Acta physiol, scand. 63: Suppl. 239, 1- 18 [1964). C arlsson, A., F alck, B., H illarp, N.-Å., T hieme, G., and T orp, A .: A new histochemical method for visualization of tissue catechol amines. Med. exp. 4: 12 3 -12 5 (19 6 1).


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C orrodi,

H., und H illarp, N.-A.: Fluoreszenzmethoden zur histochemischen Sichtbarmachung von Monoaminen, i. Identifizierung der fluoreszierenden Produkte aus Modellversuchen mit 6,7-Dimethoxyisochinolinderivaten und Formaldehyd. Helv. chim. Acta 46: 2425-2430

C19633. — Fluoreszenzmethoden zur histochemischen Sichtbarmachung von Monoaminen. 2. Identi fizierung des fluoreszierenden Produktes aus Dopamin und Formaldehyd. Helv. chim. Acta 47: 9 11-9 18 [1964). C orrodi, H., H illarp, N.-ä ., and J onsson, G .: Fluorescence methods for the histochemical demonstration of monoamines. 3. Sodium borohydride reduction of the fluorescent com pounds as a specificity test. J. Histochem. Cytochem. 12: 582-586 (1964). C sillik, B.: Histochemical model experiments on the effect of various drugs on the cate cholamine content of adrenergic nerve terminals. J. Neurochem. n : 351-355 [1964). C sillik, B., and E rulkar , S. D.: Labile stores of monoamines in the central nervous system: A histochemical study. J. Pharmacol, exp. Ther. 146: 186-193 [1964]. E ränkö , O.: On the histochemistry of the adrenal medulla of the rat, with special reference to acid phosphatase. Acta anat. 16 : Suppl. 17, 1-60 [1952). E rös, G.: Eine neue Darstellungsmethode der sogenannten “gelben” argentaffinen Zellen des Magendarmtraktes. Zbl. allg. path. 54: 385-391 (1932). F alck, B.: Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta physiol, scand. 56: Suppl 197, 1-25 [1962). F alck, B., H illarp, N.-Ä., T hieme, G., and T orp, A .: Fluorescence of catechol amines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10: 348-354 Ci962]. F alck, B., H äggendal , J., and O wman , C h.: The localization of adrenaline in adrenergic nerves in the frog. Quart. J. exp. Physiol. 48: 253-257 [1963]. F alck, B., and T orp, A .: New evidence for the localization of noradrenaline in the adrenergic nerve terminals. Med. exp. 6: 169-172 [1962). H amberger, B., N orberg, K.-A.: Histochemical demonstration of catecholamines in fresh frozen sections. J. Histochem. Cytochem. 12 : 48-49 C1 9^4^• H amberger, B., M almfors, T., N orberg, K.-A., and S achs, C h .: Uptake and accumulation of catecholamines in peripheral adrenergic neurons of reserpinized animals, studied with a histochemical method. Biochem. Pharmacol. 13: 841-844 (1964). H amperl, H .: Was sind argentaffine Zellen? Virchows Arch. path. Anat. 286: 8 11-8 33 (1932). H ess, S. M., and U denfriend , S.: A fluorimetric procedure for the measurement of tryptamine in tissues. J. Pharmacol, exp. Ther. 127: 17 5 -17 7 [1959]. H illarp, N.-A., and H ökfelt, B.: Histochemical demonstration of noradrenaline and adrenaline in the adrenal medulla. J. Histochem. Cytochem. 3: 1- 5 [1955]. H illarp, N.-A., and M almfors, T.: Reserpine and cocaine blocking of the uptake and storage mechanisms in adrenergic nerves. Life Sciences 3: 703-708 (1964]. H odgman , C. D., W east, R. C., and Selby, S. M.: Handbook of chemistry and physics, p. 2500 [i960] [Chemical Rubber Publ. Co., Cleveland]. M oline , S. W., and G lenner, G. G .: Ultrarapid tissue freezing in liquid nitrogen. J. Histochem. Cytochem. 12 : 777-783 C1964D• N eumann , K .: Gefriertrockung und Gefrieraustauschverfahren zur Fixierung histochemischer Präparate. Acta histochem. Suppl. I. 204-226 [1958]. O wman , C h ., and S jöstrand , N. O.: Short adrenergic neurons and catecholamine-containing cells in vas deferens and accessory male genital glands of different mammals. Z. Zellforsch. In press [1965]. P earse, A. G. E.: Histochemistry. Theoretical and applied., p. 25 [19 6 1] [J. & A. Churchill, London], Acta Univ. Lund. II. 1 965. No. 7 .


P earse,

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A. G. E.: Rapid freeze-drying of biological tissues with a thermoelectric unit. J. sci. Instr. 40: 176 -177 (1963). T hieme, G .: Small tissue dryers with high capacity for rapid freeze-drying. J. Histochem. Cytochem. In press (1965). W heeler, E. L.: Scientific glassblowing., p. 19 1 (1958) [Interscience Publishers Inc., LondonNew York). Y oung , M. R.: Principles and technique of fluorescence microscopy. Quart. J. micr. Sci. 102: 419-449 (4961).

Acta Univ. Lund. II. 1965 . No. 7 .

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Fig. i. Equipment for quenching tissues. A. Propane—propylene mixture is kept in a metal cylinder ( a) suspended in liquid nitrogen ( b ) in a Dewar vessel ( i litre size). B. Metal block with numbered depressions [c] for col lecting frozen tissue pieces hangs in a metal frame beneath the surface of liquid nitrogen (b) in a Dewar vessel (4 litre size). Metal hook for lifting the block (d).

Fig. 2. General view of freeze-drying apparatus. a, freeze-dryer column (for safety this is enclosed in fine-meshed metal net covered with gauze); b, inner tube ("cold finger”) ; c, two-stage mechanical pump (Edwards); d, air-cooled Acta Univ. Lund. 11 . 1965. No. 7 .


Fig. 3. Freeze-dryer column (1:8 of natural size). The outer tube a [Q.V.F., industrial pipeline, type PS 4/48) is delivered with plane flanges in both ends. One of the flanges is cut off, and the inner tube b (Pyrex glass) is welded to the outer tube at c; this work requires a large glass lathe. The surfaces between the two tubes are silvered Csee W heeler, 1958). Connexion to the vacuum line at d. The inner tube contains solid carbon dioxide (e) and acetone [/). Glass cup g (Q.V.F., type PBE/4) closes the outer tube at the flange h. Metal block CO with burr holes for the specimens lies in the bottom of the glass cup. This is immersed in the outer bath /. [The whole assembly from A.B. Kyl-Mekano, Lund, Sweden).

oil-diffusion pump [Edwards); e, air inlet; /, cold trap; g, compression-expansion unit [A.B. Kyl-Mekano, Lund, Sweden); h, cooling coil from compressor. Acta Univ. Lund. II. 1965. No. 7 .

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t'S Z Z K i

22

Fig. 4. Starting freeze-drying procedure (A -C ).

a, lower part of freeze-dryer column; b, "cold finger” ;c, thermostat thermometer for compressor; d, glass cup fitting outer tube; e, Dewar vessel [7 litre size] containing ethanol cooled by coil from compressor; f, metal block containing specimens. Acta Univ. Lund. II. 1965. No. 7 .


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Fig. 5. Dry-air perspex box for storing and handling freeze-dried specimens. a, arm ports fitted with rubber cuffs; h, dish containing phosporus pentoxide; c, metal block with burr holes containing tissue pieces; d, metal block with plastic cylinders for same purpose; e, entomologists’ forceps; f, plastic receptacles in which specimens are kept during formaldehyde treatment; g, access port.

Fig. 6. Glass container for paraffin embedding in vacuo. Vessel a containing paraffin is immersed in a water bath at + 600 C. The specimen is placed in vessel h, and after short evacuation through the outlet (c) is tipped into the paraffin by tilting the whole container. Tryckt den 22 februari 1965.
