Histochemistry 48, 269-281 (1976) Histochemistry �9 by Springer-Verlag 1976

Fluorogenic Detection of Primary Amines in Plant Histochemistry with Fluorescamine: A Comparative Study on the Effects of Coagulant and Non-Coagulant Fixatives

Alessandro Bruni, Maria P. Fasulo, Barbara Tosi, Giuseppe Dall'Olio, and Gian Luigi Vannini

Intitute of Botany, University of Ferrara, Corso Porta Mare, 2, 1-44100 Ferrara, Italy

Summary. The new highly sensitive method of fluorescamine reaction for the topochemical detection of primary amino groups was studied as a substi- tude of ninhydrin-Schiff's reaction for the localisation of total proteins in plant tissues. The influence of various coagulant and non-coagulatn fixatives on the induction of fluorescamine fluorescence was examined: ethanol, for- maldehyde gas and solution, glutaraldehyde, acrolein, osmium tetroxide, Bouin, Rossman, Clarke and Zenker's fluids and FMA were employed. It was found that the use of the fluorogenic method is conditioned by the fixative ability to keep the amino groups disposable and by its capability to reduce the natural autofluorescence of plant material. A detailed account of the fixation methodology demonstrated that non-coagulant acrolein and coagulant mercuric chloride are the most promising fixatives for the use of the fluorescamine reaction in plant histochemistry.

Introduction

Cytochemical studies on intact cells require methods capable of detecting the presence of a single component in a mixture without prior physical or chemical separation. A requirement of these studies is an extremely high sensitivity to permit the detection of small quantities of a substance present inside a single cell. Ultraviolet microscopy is, in theory, capable of such sensitivity and fluores- cent cytochemical methods offer advantages to enhance sensitivity and contrast over transmitted light absorption methods.

Recent studies have revealed the unique propensity of fluorescamine (4- phenylspiro[furan-2(3), l'-phthalan]-3,3-dione) to produce a highly fluorescent substance upon reaction with primary amines (Udenfriend et al., 1972 a, 1972b). The substance, which itself is non-fluorescent, reacts with primary amino groups to form strongly fluorescent pyrrolinones (Weigele et al., 1972). The finding provides the basis for the development of a new, sensitive reagent for the fluorometric assay of biologically important amines, including aminoacids, pep-

270 A. Bruni et al.

tides and proteins (B6hlen et al., 1973; Stein et al., 1973; De Bernardo et al., 1974; Felix and Terkelsen, 1974). The reaction efficiency, also in submicro- molar concentrations of amines, has promoted the use of fluorescamine in various practical analytical applications (Kisic and Rapport, 1974; Ranieri and McLaugblin, 1975). Moreover, the superiority of the compound with respect to the classic ninhydrin-Schiff's method in the quantitative determination has recently suggested the use of Fluram for cyto-histochemistry studies on animal section (H~tkanson et al., 1974; Larsson et al., 1975).

The present investigation has been undertaken to critically evaluate the fluo- rescent behavior of fluorescamine, when employed in the detection of amino groups in plant histochemistry in relation to tissues fixed in several coagulant and non-coagulant fixatives.

Materials and Methods

Materials. The choice of biological materials for the use in the experiments has been carried out in view of an estimation of the fluorescamine reaction in tissues at a different grade of differentia- tion and specialization. For this purpose, root tips and segments of the primary vascular cylinder of Allium eepa roots, segments of Chelidonium majus and Calystegia soldanella stems, endosperm and quiescent embryos, isolated from dry seeds of Euphorbia marginata, have been employed.

Fixation and Embedding. Both primary coagulant fixatives and non-coagulant fixatives, or fixative mixtures containing two or more primary fixatives, were used. The specimes were fixed as it follows: (a) formalin (4 g paraformaldehyde dissolved in 100 ml 0.1 M phosphate buffer, pH 7.0) for 24h; (b) 70% ethanol for 12h; (c) 10% acrolein in phosphate buffer (0.15 M, pH 7.4) at 4 ~ C for 2 h; (d) 6% glutaraldehyde in sodium cacodylate buffer (0.1 M, pH 7.0) at 4 ~ C for 2 h; (e) formaldehyde gas at 80 ~ C for 1 h obtained in a closed vessel (1,000 ml) containing 5 g paraformaldehyde previously equilibrated in air with approximately 70% humidity; (f) Rossman's fluid (95% ethanol saturated with picric acid-formalin; 9: 1) for 24 h; (g) Bouin's fluid (distilled water saturated with picric acid-formalin-glacial acetic acid; 7.5:2.5:0.5) for 48 h; (h) Zenker's fluid for 6 h (Ganter and Joll+s, 1970); (i) FMA (formalin-mercuric chloride-acetic acid; 10:6:5 in 100 ml water) for 24h (Lillie, 1954); (k) 2% osmium tetroxide at 4~ for 2 h; (1) Clarke's fluid at 4 ~ C for 12 h (Ganter and Joll+s, 1970). The single components of mixture fixatives were employed as fixatives in order to elucidate their role in the fluoreseamine reaction. To insure a better penetration, the samples were cut into slices not exceeding 3 mm in thickness. Tissues fixed in Zenker's, FMA, Bouin's and Rossman's fluid were washed overnight in running water prior to processing. All tissues were dehydrated in graded ethanol and embedded in a mixture consisting of buthyl and methyl-methacrylates 7 : 3 (Merck, A. G. Darmstadt). Using a Pyramitome LKB microtome with glass knife, 3 g sections were prepared. Sections were floated on a small drop of distilled water on a clean glass slide and warmed on a hot plate until dry to flatten them. The resin was removed by benzene before applying the histological reactions. Some specimens were collected by stripping of Allium cepa cataphytlary epiderm for fixation and stain experiments without embedding.

Cytoehemical Reactions. The fluorescamine (Fluram, Hoffman-La Roche, Basel, Switzerland) was dissolved in acetone at a concentration of 2 mg per 10 ml. The slides were placed in 0.2 M phosphate buffer, pH 8.0, for a few minutes. Immediately after removing the slides from the buffer, each section was covered with a few drops of the fluorescamine solution. After 60 sec the slides were rinsed repeatedly in phosphate buffer. Some slides for each .fixative type were stained with ninhydrin- Schiff process (Jensen, 1962) or with toluidine blue 0.1% in Walpole's acetate buffer at pH 5 for general morphology examination. After the reaction, the sections were dehydrated in graded ethanol solutions and mounted in a rapid synthetic non-fluorescent medium (Entellan, Merck).

Amines in Plants 271

Deamination. The diazo-deazo method was carried out as outlined in Pearse (1968), i.e. 48 h at 4~ in the dark in 3.3% sodium nitrate in 3% sulphuric acid. Sections were then continuously soaked for 4 h at 60 ~ C in distilled water and in absolute ethanol, subsequently subjected to histo- chemical procedure.

Controls. After dehydration, one set of slides for every fixation type was directly mounted and examined in the fluorescence microscope.

Fluorescence Microscopy. The slides were viewed with a Zeiss Photomicroscope II equipped with an incident fluorescence condenser. The light source was high-pressure mercury vapour lamp HB0 200 W/4, exciter filter as by equipment. High speed Ilford film HP4 was used for photography.

Results and Comments

The results obtained by fluorescamine reaction have been evaluated in compari- son with classic ninhydrin-Schiff 's reaction for total proteins. Reference to proteins as a substrate test has been made because in this way the observation of qualitative differences between the two methods is easier. Furthermore, it seems convenient to compare, by means of parallel tests, the sections directly fluorochromized with those previously subjected to a deamination process. In this way it has been possible to check the specificity of fluorescamine for primary amino groups. In our experiments the ninhydrin-Schiff 's reaction has always been proved qualitatively inferior to the fluorescamine reaction. Observations collected here are the result of this comparative investigation.

Non-Coagulant Fixatives

The general preservation with formaldehyde, acrolein and glutaraldehyde is excellent, though the undulations of some walls suggest some shrinkage. The image obtained in non-fluorochromized control sections shows that acrolein and glutaraldehyde possess a weak ability to make induced-fluorescence, whereas the formaldehyde solution results in a soft generalized fluorescence and accentu- ates the autofluorescence of many cellular macromolecules. The latter observa- tion is particularly true in case of permanent slides, because dehydration, embedding and mounting could change the fluorescent spectrum of cellular compounds. This is proved by strips of onion cataphyllary epiderm fixed and directely mounted in glycerine, which show a very soft autofluorescent activity when compared to the one observed in permanent slides, obtained from cut specimens. Control slides, formaldehyde gas fixed, show a good fluorescence (Fig. 1 b); this finding was expected since it is well known that this fixation type induces fluorescence with certain arylethylamines and with peptides contain- ing aromatic aminoacids in the NH2-terminal position, and moreover it results in an enhancement of the plant cell-autofluorescence (Hfikanson et al., 1971 ; Hgtkanson and Sundler, 1971; Bj6rklund et al., 1972).

After fluorescamine reaction, the specimens fixed in simple, non-coagulant fixatives have given different results. Fixation in glutaraldehyde and OsO4 pre- vents the fluorescamine-induced fluorescence, whereas the specimens fixed in

272 A. Bruni et al.

Fig. l a - d . Sections of Allium cepa root . a F luorescence induced by f luorescamine in cor t ica l paren- c h y m a t o u s cells fixed in a f o rma ldehyde solut ion, x 350. b Nucle i of p a r e n c h y m a t o u s cells. F ixa t ion by fo rma ldehyde gas w i t h o u t f luorescamine t rea tment , x 750. e F luorescence induced af ter f luoresca- mine t r ea tmen t in gaseous fo rma ldehyde fixed cells of p a r e n c h y m a t o u s tissue, x 400. d Non-f luo- rescamine f luorescent nuclei in subapica l d i f ferent ia t ing t issue of roo t fixed in fo rma ldehyde gas.

x 180

Table 1. The f luorescence po ten t i a l i ty of f lnorescamine in fixed t issues in re la t ion to the abi l i ty of the f ixat ive to p r o m o t e induced f luorescence

F ixa t ives F luorescence Decrease ( - ) or F luorescence induced by enhancemen t ( + ) induced by fixative of cel lular f luorescamine

autof luorescence

Non- Coagulant f o rma ldehyde sol. + + + fo rma ldehyde gas + + + + + +

g lu ta ra ldehyde - - - ac ro le in - - + + o s m i u m te t roxide - - -

Coagulant e thanol - + + + + + C la rke - + + + + + Bouin + + + + + R o s s m a n + + + + + Zenker - - + + F M A soft - + + +

Amines in Plants 273

Fig. 2 a-e. Appearance of cells after fixation in gaseous formaldehyde and treated with fluorescamine. a Strip of onion epiderm, x 180. b Nodal complex laticifer of Euphorbia marginata with non- fluorescent nucleus (arrow). x 280. e High fluorescence of laticifer cytoplasm in Euphorbia marginata embryo. Several corpuscles are strongly fluorescent. • 350. d Embryonic laticifers of Euphorbia marginata with several non-fluorescent nuclei, x 150. e Arrows indicate the laticifers transversally cut. High fluorescence of their cytoplasmatic content makes detection easy. x 120

aqueous fo rma l in show a g o o d abi l i ty to m a k e f luorescence in all cells, and pa r t i cu la r ly in the mer i s t emat i c and a leurone endospe rmic ones (Fig. 1 a). W i t h f luorescamine , the specimens f ixed in acrole in and fo rma ldehyde gas give s imilar results (Figs. 2 a - e and 3 a - b ) . Wi th acrole in , even t hough the f luorescence inten- sity is s l ightly less than tha t induced af ter f o r m a l d e h y d e gas f ixat ion, the con t ras t r emains very high and the reso lu t ion of the finest s t ruc tura l detai ls is possible. In the mer i s t emat ic cells, the cy top lasm, nucleus and nucleolus as well as the mi to t i c phases are well d i s t inguishable and the small vacuoles appea r to be lacking of contents . The nucleolus of comple te ly d i f ferent ia ted cells is n o r m a l l y

274 A. Bruni et al.

fluorescent with a granular, homogeneous structure; the nucleolus is frequently invisible. A particular comment must be made with respect to the internal secretory structures. The cytoplasm of laticifers, in fact, dramatically reacts to fluorescamine, the corpuscolated contents and cytomembranes become strong- ly fluorescent and make the laticifer clearly recognizable (Figs. 2c, 2e). With both fixatives in the embryonic laticifers of Euphorbia marginata, the nuclei are normally fluorescent, and especially those localized in the cotyledons and root tip react intensively to fluorescamine. Sometimes it has been noticed that many nuclei of laticifers in the lateral branches of the nodal complex, fixed in formaldehyde gas, do not react to fluorescamine (Figs. 2 b, 2 d). With formal- dehyde gas, also the nuclei of differentiating metaxylem and ryzoderm cells in root tips of Allium cepa and Calystegia soldanella are surprisingly not fluores- cent and they themselves show a negative image in a fluorescent cytoplasm (Fig. 1 d). On the contrary, the softly fluorescent nucleolus is costantly recogniz- able in the nuclear matrix. The same aspect, but with a lesser frequency, is observable in differentiating parenchymatous medullar and cortical cells. These contradictory aspects of the fluorescamine-nuclei reaction in gas formaldehyde fixed tissues, also observed by Hgtkanson et al. (1974) in several cells of animal tissues, cannot be understood in the light of the present information.

Coagulant Fixative

In the class of coagulant fixative, we have included all those fixatives, used singly or in a mixture, possessing a coagulant effect on proteins. The detailed understanding of the fixative action of mixture solutions is very difficult because it is impossible to discriminate the concurrent action of each component of the mixture. For this reason, we have used both material fixed in a mixture and material fixed in each fixative agent of a mixture.

In the control sections fixed in ethanol and Clarke's fluid the wall, nucleus and nucleolus are clearly distinguishable because of their well-known auto fluores- cence. After fluorescamine treatment, the results are topologically and substan- tially identical for both fixatives: strong generalized fluorescence and soft con- trast. Fixatives containing ethanol, in fact, act on proteins and change their macromolecular structure with no direct reaction on amino groups. This means that, after fixation, the presence of free amino groups disposable for fiuoresca- mine reaction is not a sufficient reason to give a well-contrasted optical image.

The results obtained in specimens fixed by Bouin's and Rossman's fluids are analogous. The non-fluorescamine treated sections exhibit a moderate auto- fluorescence, most likely induced both by the picric acid-protein complex and by fixative traces in the ground protoplasm. The fluorescence that fluorescamine induced on tissues placed in these fixatives is good, both in intensity and contrast. Furthermore, while the nuclei and chromosomes appear rather well preserved, the nucleolus is somewhat shrunken. Because of several secondary effects produced by picric acid in ultraviolet light, the results obtained by Bouin's and Rossman's fixative mixtures frequently present a lower quality with respect to that obtained with non-coagulant fixatives.

Amines in Plants 275

Fig. 3. a Fluorescence induced by fluorescamine in strip of onion epiderm after acrolein fixation. x 250. b Nucleus of Calystegia soldanella parenchymatous cells. Fixation by acrolein and fluoresca- mine treatment, x 1,600. e Meristematic cells of Allium cepa root. Fixation by Zenker's fluid and fluorescamine treatment. The cytoplasmatic damage is consistent; nuclei are well preserved. x 330. d Parenchimatous cells of onion root fixed in FMA fluid without fluorescamine treatment. The fluorescence is reduced, x 300

All control specimens placed in Zenker ' s and F M A fixative mixtures show a very weak fluorescence and the reduced and evanescent autofluorescence is located in the cell wall (Fig. 3 d). The plant material killed with the mercuric fixatives and f luorochromized by fluorescamine shows a fluorescence of good intensity with excellent contras t (Fig. 3 c, Figs. 4a-c) . These advantages are asso- ciated with a satisfactory resistence to fading and a weak ability to induce autoflorescence. Fluorescence induced by fluorescamine in plant tissues fixed by HgC12 solution confirms that the results obtained with Zenker 's and F M A fluids are essentially due to the mercuric chloride act ion on the macromolecula r structure o f proteins. The F M A fixative mixture differs f rom Zenker ' s fluid for the presence o f formaldehyde as substitute o f potass ium dichromate. This

276 A. Brnni et al.

Fig. 4a--e. Tissues of Allium cepa fixed in FMA fluid and treated with fluorescamine, a-b Sections of meristematic cells of root. After FMA fixation the contrast is very high. x 350, x 450. c Strip of onion epiderm. • 500

substitution produces a more delicate and less oxidative fixative action. The fluorescent staining quality of fluorescamine is similar both in tissues fixed by F M A fluid and in tissues fixed by gaseous formaldehyde, although the intensity of fluorescence is higher with the former fixative mixture. The general pattern of the fluorescence induced by FMA-fluorescamine is particularly clear in samples of meristematic cells. With this fixative, the metaphase and anaphase chromosomes are well visible, the mitotic spindle appears fibrous, but unfortu- nately the nuclear sap becomes coarsely granular and the nucleolus is often swollen. Non-fluorescent nuclei are never present. The laticifer nuclei show good fluorescence, also in the lateral branches of the nodal complex of Euphorbia marginata embryos, but the cytoplasm is collapsed and devoid of corpuscles.

Amines in Plants 277

Aleurone granules are strongly fluorescent in the endospermic cells of Euphorbia marginata seeds.

The good qualities shown by mercuric fixatives in ultraviolet microscopy are in contrast with the evident damage caused by their excessively drastic coagulant action, especially at the cytoplasmic level.

Discussion

Fluorescamine is a higly sensitive reagent for the detection of primary amino groups, but its use as a substitute for ninhydrin-Schiff's reaction in plant his- tochemistry is strongly conditioned by the ability of the fixative to keep the amino groups disposable. According to previous reports, fluorescamine readily produces fluorescence upon reaction with the following chemical groups: (a) NH2-terminus of peptides and proteins; (b) s-amino group of lysine; (c) amino sugar of glycoproteins; (d) amino-phospholipids of lipoproteins (Weigele et al., 1972; B6hlen et al., 1973; Ragland et al., 1973; Stein et al., 1973a; Stein et al., 1973b; Hgtkanson et al., 1974; Kisic and Rapport, 1974). Therefore, the formation of fluorescent pyrrolinones on tissue sections chiefly occurs with those proteins, peptides and amino sugars, which have not been extracted during the slide preparation. For this reason, we believe that in this case the extracting effect caused by the washing solution, dehydration and rehydration takes on an importance which is not to be underestimated in improving the efficiency of fluorescamine reaction. Results of this study indicate that it is extremely important to select the fixative keeping in mind the chemical characteristics of the compound to be tested and noting the reactivity of this chemical with the fixative.

Our studies have confirmed that fluorescamine-induced fluorescence is prevented by fixation in glutaraldehyde and osmium tetroxide. This was expected since the glutaraldehyde fixation of proteins introduces both intra- and intermo- lecular crosslinks especially with free amino groups of proteins (Bowes and Cater, 1966; Richards and Knowles, 1968). The glutaraldehyde-protein complex is irreversible, since as it is known, the crosslinks introduced by glutaraldehyde are very stable (Cater, 1963 ; Hopwood, 1972). Furthermore, Hughes and Thur- man (1970) reported that the s-amino groups also react strongly with glutaralde- hyde. Consequently, there is no evidence to suggest the use of glutaraldehyde as a fixative in amino group detection. Although scanty information is available on the interaction between osmium tetroxide and proteins, some evidence indi- cates that oxidative deamination of osmicated tissue proteins is not uncommon (Adams et aI., 1967; Needles, 1967; Burkl and Schiechl, 1968; Millonig and Marinozzi, I968; Hopwood, 1969; Hopwood, 1970). Owing to this fact, it seems obvious that in osmicated tissues, the use of fluorescamine is to be discouraged. Nevertheless, the experiments conducted seem to confirm the high fluorescamine specificity for amino groups.

Using formaldehyde-fixed material, we have found that fluorescamine in- duces fluorescence in several cell systems. This was unexpected since the fixative reaction of this monoaldehyde with proteins involves the free amino groups

278 A. Bruni et al.

with formation of amino methylol groups, which thereafter condense with other functional groups such as phenol, imidazole and indole to form methylene bridges (Lojda, 1965 ;. Hopwood, 1969). After formaldehyde fixation, a conceiv- able explication-of the presence in protein chains of primary amino groups which are disposable to fluorescamine reaction can be given by the binding reversibility between formaldehyde and free amino groups. Besides, it is known that since the formaldehyde molecule is relatively small, many reactive groups of the tissue escape the formation of bridging links (Bowes et al., 1965). If so, after formaldehyde fixation, some amino groups could remain available for fluorescamine reaction. Consequently, because of the presence of weak cross- links introduced into the tissue by formaldehyde, the swiftness of washing and dehydration of sections become a determinating factor in the efficiency of fluorescamine-protein reaction.

It is known that acrolein fixation shows a high specificity for proteins; this aldehyde, in fact, rapidly reacts with sulphydryl groups, but the reaction leading to protein-bound aldehyde groups is also due to other groups including the amino groups (Van Duijn, 196l). In spite of these premises, after acrolein fixation primary amino groups remain available and react with fluorescamine causing a strong fluorescence of the specimens. A possible explanation of these unexpected results is given by the probable existence of "buried" amino groups which do not react with acrolein perhaps because of steric hindrances. This suggests the existence of amino groups that are in some way "protected" by acrolein.

Among the non-coagulant fixatives employed, gaseous formaldehyde and acrolein have provided the best results for contrast and intensity of fluorescence. In fluorescent cytochemical methods for plant tissues, however, formaldehyde gas meets several difficulties in application as a fixative on account of the ability to increase the background fluorescence of the specimen. In order to overcome these difficulties and to distinguish with clearness the fluorescamine- induced fluorescence over the fixative-induced one, Hgtkanson et al. (1974) have proposed some methodological proceedings which make this distinction clear. They are: speed of fading and regeneration of fluorescamine-induced fluores- cence; weakness of formaldehyde-induced fluorescence and difference between spectral characteristics of fluorescence. Unfortunately these operative proceed- ings are of limited utility in cytochemistry because of the high contents in plant cells of compounds whose autofluorescence is increased by formaldehyde gas. The interpretation of the optical image appearing to the observer after gas formaldehyde fixation and fluorescamine treatment can be very complicated depending upon the type of tissue. Formaldehyde gas, therefore, limits the critical ability of cytologists. On the contrary, with acrolein the inconveniences are absent since this aldehyde does not promote the fluorescence and depresses the natural autofluorescence of many plant tissues. A general hindrance to the use of aldehydic fixatives, however, is the fast fading of the tissues fixed with these compounds. For this reason, the observation by a microscope equipped with an incident illumination apparatus is strongly suggested.

In material killed by ethanol based fixatives, the fluorescamine topochemical detection of amino groups gives poor quality results because of overall fluores-

Amines in Plants 279

cence and weak contrast. Other coagulant fixative mixtures such as Bouin's and Rossman's, furnish non-appreciable results, also because of the particular chemical and physical characteristics of picric acid. In plant tissues, this com- pound produces an enhancement of overall fluorescence and acting as an undesir- able fluorogenic agent causes a deterioration of the optical image. These hin- drances greatly restrain the use of fixative mixtures containing picric acid in the amino group localization by fluorescamine.

On the basis of the obtained results, the coagulant fixatives suitable to be used in the fluorescamine reaction are those with mercuric chloride. The reaction of mercuric chloride with proteins has been carefully studied by several authors (Hughes, 1947; Gomori, 1956; Hopwood, 1972). The mercury-protein reaction was found to be strongly affected by acidity and alkalinity. In F MA and Zenker's fixative mixtures, mercuric chloride is in a highly acid medium in which the compound reacts with sulphydril groups of cysteine only (Gomori, 1956). In this way the mercury forms a link between cysteine side-groups in neighbouring protein chains and it leaves the amino groups available for fluo- rescamine reaction.

Experience gained so far indicates that the results obtained with mercuric coagulant fixatives can be considerably improved especially by an adequate dosing of the fixative mixture in order to limit the cytoplasmic damage caused by its drastic coagulant action.

In conclusion, under the conditions developed in this investigation, the fluo- rescamine reaction is particularly suited for plant histochemical proceedings, but it is difficult to indicate a fixative of general employment which satisfactorily suits all analytical needs. No one fixative is ideal for all situations. By comparing the results and assessing the loss of substances from the tissue during processing, it is possible to circumscribe the fixative choice. For the employment of fluoresca- mine reaction, it is convenient to use the fixatives which present reduced second- ary fluorescent effects and leave the amino groups free. The results, therefore, show the importance of the conditions of fixation of tissues in fluorescence studies and indicate that the mercuric fixatives can produce a considerable increase in the intensity of fluorescamine-induced fluorescence for cellular de- monstration of amines in plant histochemistry.

References

Adams, C.W.M., Abdulla, Y.H., Bayliss, O.B. : Osmium tetroxide as a histochemical and histological reagent. Histochemie 9, 68-77 (1967)

Bernardo, S. de, Weigele, M., Toome, V., Manhart, K., Leimgruber, W., B6hlen, P., Stein, S., Udenfriend, S.: Studies on the reaction of fluorescamine with primary amines. Arch. Biochem. Biophys. 163, 390 399 (1974)

Bj6rklund, A., Falck, B., Owman, Ch. : Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In: Methods of investigative and diagnostic endocrinology, (S.A. Berson, ed.), Vol. 1: The thyroid and biogenic amines (J.E. Rall and I.J.Kopin, ed.), pp. 318-368. Amsterdam: North-Holland PuN. Comp. 1972

B6hlen, P., Stein, S., Dairman, W., Udenfriend, S. : Fluorometric assay of proteins in the nanogram range. Arch. Biochem. Biophys. 155, 213-220 (1973)

280 A. Bruni et al.

Bowes, J.A., Cater, C.W.: Crosslinking of collagen. J. appl. Chem. (Lond.) 15, 296-304 (1965) Bowes, J.A., Cater, C.W. : The reaction of glutaraldehyde with proteins and other biological mate-

rials. J. roy. micr. Soc. 85, 193-200 (1966) Burkl, W., Schiechl, H.: A study of osmium tetroxide fixation. J. Histochem. Cytochem. 16,

157-161 (1968) Cater, C.W. : The evaluation of aldehydes and other bifunctional compounds as crosslinking agents

for collagen. J. Soc. Leather Trades Chemists 47, 259 (1963) Duijn, P. van: Acrolein-Schiff, a new staining method for proteins. J. Histochem. Cytochem.

9, 234-241 (1961) Felix, A.M., TerkeIsen, G.: Fluorometric analysis of Ne-methylamino-acids using fluorescamine.

Analyt. Biochem. 60, 78-87 (1974) Ganter, P., Joll6s, G.: Histochimie normale et pathologique, Vol. I e II. Paris: Gauthier-Villars

1970 Gomori, G. : Histochemical methods for protein-bound sulphydryl and disulphide groups. Amer.

J. micr. Sci. 97, 1-9 (1956) Hgtkanson, R., Larsson, L.I., Sundler, F.: Fluorescamine: A novel reagent for the histochemicaI

detection of amino groups. Histochemistry 39, 15-23 (1974) HSkanson, R., Sj6berg, A., Sundler, F.: Formaldehyde induced fluorescence of peptides with N-

terminal 3,4-Dihydroxyphenylalanine or 5-Hydroxytryptophan. Histochemie 28, 36%371 (1971 a) HS.kanson, R., Sundler, F.: Formaldehyde condensation. A method for fluorescence microscopic

demonstration of peptides with NH2-terminal tryptophan residues. J. Histochem. Cytochem. 19, 477-482 (1971 b)

Hopwood, D.: The reactions between formaldehyde, glutaraldehyde and osmium tetroxide and their fixation effects on bovine serum albumin and on tissue blocks. Histochemie 24, 56-64 (1970)

Hopwood, D. : Fixatives and fixation: a review. Histochem. J. 1, 323-360 (1969) Hopwood, D. : Theoretical and practical aspects of glutaraldehyde fixation_ Histochem. J. 4, 267-303

(1972) Hughes, W.L.: An albumin fraction isolated from human plasma as a crystalline mercuric salt.

J. Amer. chem. Soc. 69, 1836 1837 (1947) Hughes, R.C., Thurman, P.F.: Crosslinking of bacterial cell walls with glutaraldehyde. Biochem.

J. 119, 925-926 (1970) Jensen, W.A. : Botanical histochemistry. San Francisco and London: W.H. Freeman C. 1962 Kisic, A., Rapport, M.M. : Determination of long-chain base in glycosphingolipids with fluoresca-

mine. J. Lipid Res. 15, 179 181 (1974) Larsson, L.I., Sundler, F., Hgtkanson, R. : Fluorescamine as a histochemical reagent: Demonstration

of polypeptide hormone-secreting cells. Histochemistry 44, 245-253 (1975) Lillie, R.D. : Histopathologic technic and practical histochemistry. New York: The Bla;kiston Co.

1954 Lojda, Z. : Fixation in histochemistry. Folia morph. (Warszawa) 13, 65-83 (1965) Millonig, G., Marinozzi, V.: Fixation and embedding in electron microscopy. In: Advances in

optical and electron microscopy (Barer, R. and Cosslett, V.E., eds.), Vol. 2, p. 251. New York: Academic Press 1968

Needles, H.L. : Crosslinking of gelatin by aqueous peroxydisulfate. J. Polym. Sci. Ag, 1-13 (1967) Pearse, A.G.E. : Histochemistry. Theoretical and applied, Vol. I, Third ed. London: J. & A. Churchill

Ltd. 1968 Ragland, W.L., Pace, J.L., Kemper, D.L.: Fluorescence scanning of proteins in polyacrilamide

gels. I.R.C.S. (73-7), 3-2-6 (1973) Ranieri, R.L., McLaughlin, J.L.: Cactus alkaloids: XXVII. Use of fluorescamine as a thin-layer

chromatographic visualization reagent for alkaloids. J. Chromatogr. 3, 234-238 (1975) Richards, F.M., Knowles, J.R.: Glutaraldehyde as a protein crosslinking agent. J. molec. Biol.

37, 231-233 (1968) Stein, S., B6hlen, P., Imai, K., Stone, J., Udenfriend, S.: Assay of amino acids, peptides, proteins

and other primary amines with fluorescamine. Fluorescence News 7, 9-10 (1973a) Stein, S., B6hlen, P., Stone, J., Dairman, W., Udenfriend, S. : Amino acid analysis with fluorescamine

at the picomole level. Arch. Biochem. Biophys. 155, 202-213 (1973b) Udenfriend, S., Stein, S., B6hlen, P., Dairman, W.: A new fluorometric procedure for assay of

Amines in Plants 281

amino acids, peptides and proteins in the picomole range. Third American Peptide Symposium, Boston, June 1972a, J. Meienhofer, ed. (Ann. Arbor-Humphrey Science, Ann. Arbor, Michigan)

Udenlu S., Stein, S., B6hlen, P., Dairman, W., Leimgruber, W., Weigele, M. : Fluorescamine: a reagent for assay of amino acids, peptides, proteins and primary amines in the picomole range. Science 178, 871-872 (1972b)

Weigele, M., De Bernardo, S.L., Tengi, J.P., Leimgruber, W. : A novel reagent for the fluorometric assay of primary amines. J. Amer. chem. Soc. 95, 5927-5928 (1972)

Received April 3, 1976