Histochemistry60, 113- 123 (I979)

Histochemistry 9 by Springer-Verlag 1979

Aldehyde-Fuchsin: Historical and Chemical Considerations Holde Puchtler, Susan N. Meloan, and Faye Sweat Waldrop Department of Pathology,MedicalCollegeof Georgia, Augusta, Georgia30901, USA

Summary. The staining mechanisms of Gomori's aldehyde-fuchsin are not yet fully understood. It seemed therefore timely to review the history of this dye class in context with current dye and aldehyde chemistry. In 1861 Lauth treated basic fuchsin with acetaldehyde. This dye became known as Aldehyde Blue, but consisted of violet and blue dyes. Schiff (1866) studied several aldehyde-fuchsins; these compounds contained two molecules of dye and three molecules of aldehyde. Acetaldehyde-fuchsin prepared according to Schiff's directions showed staining properties similar to those of Gomori's aldehyde-fuchsin. This dye class was soon superseded by new dyes more suitable for textile dyeing, and chemical investigations of aldehyde-fuchsins ceased around the turn of the century. Gomori's aldehyde-fuchsin has been regarded as a Schiff base. However, according to chemical data, low molecular aliphatic aldehydes and aromatic amines tend to form condensation products. Correlations of chemical and histochemical observations suggest such processes during aging of dye solutions. Models of dimers and polymers of aldehyde-fuchsin could be built without steric hindrance. The nature of the bonds formed by various components of aldehyde-fuchsin solutions is not clear. However, cystine in proteins, e.g. in basement membranes, apparently does not play a role in the binding of aldehyde-fuchsin by unoxidized Carnoy- or methacarn-fixed sections. Introduction In a brief note Gomori (1950) introduced aldehyde-fuchsin as a stain for elastic fibers; beta cells in islets of pancreas, certain basophils in pituitary, and some kinds of mucin were also colored. This stain was studied intensively, e.g. by Scott and Clayton (1953), Halmi and Davies (1953), Bangle (1954), Sumner (1965) and von Denffer (1973). Nevertheless, the chemical mechanisms of the aldehyde-fuchsin stain are not yet clear. A review of the extensive literature is beyond the scope of this report.

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Parenthetically, though the staining properties of aldehyde-fuchsin received much attention, the raison d'6tre of this dye seems to be little known. As G o m o r i told one of us (H.P.) in 1953, he was interested in the controversy concerning the Feulgen reaction which ensued from the work of Stedman and others (see review by Pearse, 1968). G o m o r i therefore prepared compounds of various aldehydes and basic fuchsin. Paraldehyde-fuchsin did not stain nuclei, but colored elastic tissue so nicely that he published it as a stain for the latter. However, G o m o r i was not particularly interested in elastic tissue and, in 1953, did not intend to study aldehyde-fuchsin any further. Clearly, the aldehydefuchsin stain owes its existence to serendipity. N u m e r o u s studies demonstrated reactions of aldehyde-fuchsin with a variety of groups in tissues. Von Denffer (1973) suggested that there were at least five types of bonds; 1) ionic bonds with acid groups; 2) covalent bonds with sulfonic acid groups; 3) covalent bonds with carbonyl groups; 4) covalent bonds with tautomeric enamines, e.g. in oxytalan fibers; 5) non-ionic bonds. D a t a by Goldstein (1962) indicated formation of hydrogen bonds with elastic tissue. However, it is difficult to explain the diverse staining mechanism of aldehydefuchsin on the basis of its conventional formula of a Schiff base (Harms, 1958). Since aldehyde-fuchsins were studied intensively already in the 1860's, it was deemed of interest to review early data and to re-evaluate the structural formula of aldehyde-fuchsin on the basis of current dye and aldehyde chemistry. Problems of staining of various tissue structures have been dealt with in several comprehensive reviews (Sumner, 1965; von Denffer, 1973; Mowry, 1978) and were therefore omitted from this study. The term elastin will be used to denote structures with the histochemical and fluorescence microscopic properties of purified elastin. Fibers which show the histochemical and fluorescence microscopic properties of collagens and exhibit affinity for aldehyde-fuchsin and other so-called elastica stains will be referred to as pseudo-elastica. For m a n y years G o m o r i ' s aldehyde-fuchsin has yielded easily reproducible staining patterns in our hands and was used exclusively in this study.

Materials and Methods Experiments were carried out on human autopsy material. Blocks of aorta, kidney, lung and salivary glands were fixed in methacarn solution and embedded in Paraplast. Sections were cut at 5 gm. Gomori's (1950), Schiff's (1866) and Gattermann's and Wichmann's (1889) aldehyde-fuchsin solutions were prepared as recommended by these authors. In studies of effects of aging on staining properties of solutions of Gomori's (1950) aldehyde-fuchsin, samples ranging in age from a few minutes to 22 months were employed. In order to distinguish between effects of substitution of amino groups in monomolecular dyes and probable cross-linking of dye molecules in aldehyde-fuchsins, Pararosanilin (C.I. 42500), Ethyl Violet (C.I. 42600) and Dahlia (C.I. 42530) were employed in 70% ethanol plus 1 ml of concentrated HCI per 100 ml of dye solution, i.e. the solvent of Gomori's aldehyde-fuchsin. Aqueous solutions of these dyes and of aldehyde-fuchsin were also studied. Other series were pretreated with periodic acid or the periodic acid-sodium bisulfite sequence and then stained. The periodic acid-Schiff (PAS) and the periodic acid-sodium bisulfite-resorcin-

Aldehyde-Fuchsin

!15

fuchsin (PBRF) reaction (Puchtler and Sweat, 1964a) respectivelywere used as standards in the evaluation of staining patterns. Earlier experiments on effects of modification of reactive groups in tissues on the binding of aldehyde-fuchsinwere reviewed. These studies were performed during investigations of the resorcin-fuchsin stain (Puchtler et al., 1961; Puchtler and Sweat, 1963, 1964b), but not published. To detect possibleeffectsof steric hindrarme, models of various potential Pararosanilin-acetaldehyde reaction products were built with atomic models of the Briegleb-Stuarttype. Results

Comparison of Different Dyes." Pertinent staining properties of Pararosanilin, Dahlia, Ethyl Violet and aldehyde-fuchsin are summarized in Table 1. Affinity of the first three dyes for tissue structures decreased with increasing substitution of amino groups of Pararosanilin. Aldehyde-fuchsin showed different staining patterns which cannot be explained by formation of simple Schiff bases at one or two amino groups of the dye, nor by an increase in basicity. Effects of Age. Effects of age of aldehyde-fuchsin solutions on dye binding are shown in Table 2. The change of color within 30 rain after preparation of the dye solution indicates substitution of amino groups of Pararosanilin. However, the alterations of staining properties at 48 h and later cannot be accounted for by formation of Schiff bases according to the currently accepted formula (Harms, 1958), but suggest additional or different processes in aldehydefuchsin solutions.

Pretreatment with Periodic Acid or Periodic Acid and Sodium Bisulfite. After oxidation with periodic acid, aldehyde-fuchsin stained elastin intensely, but showed little or no reaction with basement membranes. Vice versa, Schifl's reagent colored basement membranes intensely, but left elastin unstained. Pseudoelastic collagen reacted nicely with both procedures. The staining patterns of Pararosanilin resembled those of Schiff's reagent. Ethyl violet stained elastin strongly, but coloration of other tissue structures was too patchy for critical evaluation. In sections pretreated with periodic acid and sodium bisulfite, resorcinfuchsin, aldehyde-fuchsin and Pararosanilin colored basement membranes, pseudo-elastic fibers and elastin intensely. Reticulum fibers and collagen reacted moderately to strongly with aldehyde-fuchsin, but were left unstained by Pararosanilin and resorcin-fuchsin. Nuclei stained intensely with Pararosanilin, but did not bind aldehyde-fuchsin nor resorcin-fuchsin. Binding of Ethyl Violet tended to be patchy and was therefore not evaluated.

Comparison of Different Aldehyde-Fuchsins. Schiff's (1866) and Gomori's (1950) aldehyde-fuchsin solutions showed similar staining properties, but coloration of elastin and pseudo-elastica was less intense with Schiff's solution. In the procedure by Gattermann and Wichmann (1889) aldehyde-fuchsin is dissolved in water. Such solutions were unstable. Because of extensive precipitation of the dye, staining patterns could not be evaluated. To avoid duplication, other findings are included in the discussion below.

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Table 1. Comparison of staining properties of Pararosanilin, its derivatives and aldehyde-fuehsin Dye

Elastin

Pseudoelastica

Other collagens

Nuclei

Pararosanilin Dahlia Ethyl Violet Aldehyde-fuchsin

2-4 _+ 3 _+ 5

13 0 1 0-1 5

0-2 0-_+ 0-+ 0

3-5 24 1-4 0

Table 2. Effects of age on staining properties of aldehyde-fuchsin solutions Age of solution

Elastin

Schwalbe's sheaths

Pseudoelastica

Mucus (bronchi)

Nuclei

0 to 15 rain 30 min 48 h 2.5 months 6 months 14 months 22 months

0-_+ 4-5 5 5 4-5 4 5 4

0-+ 4-5 5 5 5 5 5

0-_+ 4-5 5 5 5 5 5

0-_+ 0 5 3-5 2-4 2-3 1-2

1-2' 2-3"* 0 0 0 0 0

Color of stain : * =pink, ** =violet

Discussion

History of Aldehyde-Fuchsins L a u t h (1861, original not available) prepared aldehyde-fuchsin f r o m basic fuchsin and acetaldehyde (Bolley and K o p p , 1897; Magnus, 1910; V e n k a t a r a m a n , 1952). This dye was termed " A l d e h y d b l a u " (Schiff, 1866; G a t t e r m a n n and W i c h m a n n , 1889); others described the color as violet (Schubert, 1866; Nietzki, 1906; Magnus, 1910). A c c o r d i n g to Schiff (1865), it was a mixture of violet and blue dyes. Aldehyde Blue dyed textile fibers very nicely, but the fastness properties were not satisfactory. U p o n the advice o f a photographer, Cherpin a r o u n d 1861 tried to " f i x " the dye with sodium thiosulfate; this treatment converted Aldehyde Blue to Aldehyde Green, the first synthetic green dye (Gatt e r m a n n and W i c h m a n n , 1889; Nietzki, 1906). Both dyes were k n o w n to histologists (Magnus, 1910). Parenthetically, as discussed previously (Puchtler et al., 1975), the structure of dyes o f the basic fuchsin g r o u p was u n k n o w n until E. and O. Fischer (1878) identified t h e m as derivatives of triphenylmethane. Furthermore, the early termin o l o g y does not always coincide with current nomenclature. Therefore, unless

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chemical data permit identification of the dye employed, the term basic fuchsin will be used. Schiff (1866) studied various aldehyde-fuchsins in detail. Upon reaction with aldehydes the color of basic fuchsin changed gradualy from red to violet and blue ; this reaction was facilitated (,,gef6rdert") by addition of HC1 and alcohol. In comparative studies of other bases, toluidine reacted less readily than aniline, but the reaction products were analogous. Perhaps Rosanilin, which contains one o-toluidine ring, also reacts more sluggishly than Pararosanilin, which lacks the methyl group. This would explain reports that Rosanilin is unsuitable for preparation of Gomori's aldehyde-fuchsin (Mowry, 1978). Magenta II and New Fuchsin should be even less satisfactory. Schiff (1866) found that aldehydes reacted always with two molecules of a monoamine which were, so to speak, soldered together by the substitution (" ... dab die Aldehyde stets auf zwei Molectile eines Monoamins einwirken und diese durch die Substitution gleichsam zusammengel6tet werden"). Similar results were obtained with basic fuchsin. The reaction of basic fuchsin and acetaldehyde was assigned the formula 2 C2oH16 2 C2oH19N3 + 3 C2H40

9

N6

+ 3 H20 3 C2H4

i.e. Schiff's (1866) aldehyde-fuchsin consisted of two molecules of the dye and three molecules of acetaldehyde. This formula differs significantly from the currently accepted structure (Harms, 1958) which consists of one dye molecule plus one or two aldehyde residues. However, the staining properties of Schiff's (1866) aldehyde-fuchsin proved to be similar to those of Gomori's aldehydefuchsin, at least in methacarnfixed tissues employed in this investigation. In later studies Miller and P16chl (1891) described a reaction product of Pararosanilin and acetaldehyde which contained six molecules of aldehyde per dye molecule and suggested the formula C --(C6H 4 --N = CH --CH2 --CHOH -- CH3)3 Formation of this compound was supposed to occur readily at low temperatures (Meyer and Gnehm, 1897). Nietzki (1906) expressed doubts concerning this formula and regarded the violet acetaldehyde-basic fuchsin compounds as analogs of alkylated basic fuchsins. During the latter part of the 19th century Aldehyde Blue was replaced by dyes more suitable for textile dyeing and chemical studies of reactions of basic fuchsin with various aldehydes apparently ceased.

Reactions of Aliphatic Aldehydes with Amines The Schiff base formula of aldehyde-fuchsin does not provide an explanation of the striking differences between staining properties of aldehyde-fuchsin and Ethyl Violet or other alkylated Pararosanilin derivatives. According to chemical data, stable Schiff bases are formed mainly by aromatic aldehydes; those formed

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Fig. 1. Model of aldehyde-fuchsindimer. Two moleculesof Pararosanilin are linked by acetaldehyde. Schiff's (1866) aldehyde-fuchsincontainedtwo additional aldehyderesidues. Reaction of remaining free amino groups with acetaldehydecan lead to chain formation

by aliphatic aldehydes are often subject to condensation (McOmie, 1973). Polymerization of Schiff bases derived from low molecular aldehydes was demonstrated by Wagner (1954). That acetaldehyde indeed forms polymerization products is obvious from its use in the production of resins (Hadley, 1973). Since aldehyde groups of formaldehyde and acetaldehyde give analogous reactions (Hiickel, 1941), equations used for one of these aldehydes usually can be applied to both. However, acetaldehyde reacts much less vigorously than formaldehyde, e.g. in the tanning of leather (Gustavson, 1956; Walker, 1964). This difference is probably due to induction and charge effects of the alkyl group in acetaldehyde (Walker, 1964). Low molecular aliphatic aldehydes react with unsubstitued amino groups and form a variety of compounds (Wagner, 1954; Gustavson, 1956; Walker, 1964; Rath, 1972). According to these chemical data, the following reactions presumably occur in solutions of aldehyde-fuchsin (P = Pararosanilin) : P - N H 2 + O C H R -~ P - N H - C H R - O H P - N H - C H R - O H + H g N - P' --* P - N H - C H R - H N - P' + H 2 0 Such dimers of pararosanilin and acetaldehyde can easily be constructed with atomic models (Fig. 1). The remaining --NH2 groups of the dye molecules can also react with aldehydes; thus formation of chain-like polymers of Pararosanilin and acetaldehyde would not pose any steric problems. Parenthetically, Schiff's (1866) aldehyde-fuchsin can easily be built by adding two acetaldehyde molecules to free amino groups; ring closure via an aldehyde bridge is feasible, but involves some straining of bonds. Other Schiff base dimers obtained from aniline and acetaldehyde in cold alcohol in the presence of acid were regarded as Eckstein bases (Wagner, 1954), e.g.

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6H5

H2C

CH2

I

Fig. 2. Structural formula of cyclic trimer of aniline and formaldehyde (Walker, 1964). According to Wagner (1954), analogous trimers are formed by various aromatic amines and low molecular aliphatic aldehydes

I

%Hs--N

N--C6H5 H2

Fig. 3. Model of a trimer of aldehyde-fuchsin. Three molecules each of Pararosanilin and acetaldehyde are combined according to the formuta in Fig. 2. Conceivably, additional aldehyde molecules can react with any of the six free amino groups to form polymers

CH3--CH--CH2--CH=N--C6H s

I

NH--C6H5 T h e a n a l o g o u s P a r a r o s a n i l i n - a c e t a l d e h y d e c o m p o u n d has f o u r u n s u b s t i t u t e d a m i n o g r o u p s , h e n c e f u r t h e r r e a c t i o n s a n d f o r m a t i o n o f p o l y m e r s a p p e a r s possible.

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Reaction products of various aromatic primary amines with aliphatic aldehydes are known only as polymers, which may be either sharply defined cyclic trimers or indefinite linear aggregates (Wagner, 1954). Figure 2 shows the struc tural formula of the cyclic trimer of aniline and formaldehyde (Walker, 1964)_ The analogous trimer of Pararosanilin and acetaldehyde can be built without steric hindrance or straining of bonds (Fi.g 3), i.e. such compounds are at least theoretically feasible. The remaining free amino groups of the dyes may perhaps react with further aldehyde molecules. Clearly, on the basis of chemical considerations, one may expect a variety of compounds in aldehyde-fuchsin solutions. However, the exact composition of aldehyde-fuchsin solutions and their changes with age can be determined only by physico-chemical methods. Such facilities are not available to us.

Correlation of Chemical Data and Histochemical Observations Spectrophotometric investigations demonstrated considerable alterations of aldehyde-fuchsin during aging (Ortman etal., 1966). Chromatographic studies showed up to eleven compounds (v. Denffer and Heidbrink, 1974). The relations between substitution and color of triaminotriphenylmethane dyes are well under~ stood (Prud'homme, 1896; Venkataraman, 1952; Griffiths, 1976). The continual reaction of dye molecules with acetaldehyde causes also progressive changes in staining properties, e.g. decrease or loss of affinity for granules in beta cells of pancreas within the first month (Ortman et al., 1966), and a more gradual decrease in affinity for bronchial mucins and e[astin (Table 2). That these alterations are not due to increasing substitution of amino groups, without cross-linking of dye molecules, is indicated by the quite different staining patterns of Ethyl Violet (hexaethyl-Pararosanilin) and Dahlia, a mixture of tetra-, penta and hexamethylated Pararosanilins (Table 1). Extensive cross-linking of Pararosanilin by aldehyde is apparent in experiments with the vigorously reacting formaldehyde. Within 30 min Pararosanilin formaldehyde solutions are transformed into dark blue-violet precipitates and a pink solution with the staining properties of Pararosanilin. The much less reactive acetaldehyde is slowly released from paraldehyde, hence interaction with dye molecules proceeds gradually. Cross-linking of Pararosanilin molecules by acetaldehyde should cause little or no electronic interaction between dye molecules because -Ctt2- and similar groups act as insulators (Venkataraman, 1952). This lack of electronic coupling is indicated also by the similarity of color of "ripened" aldehyde-fuchsin, Ethyl and Crystal Violet. Pararosanilin derivatives with conjugated aromatic substituents are blue, e.g. Spirit Blue (C.I. 42775). In aldehyde-fuchsin each component dye molecule retains its positive charge, but binding by tissue components is determined also by steric factors and by the solvent. The importance of solvents and dye configurations in the staining of elastin and collagens has been demonstrated in studies of sulfonated dyes (Younker et al., 1978) and of cationic dyes (Meloan and Puchtler, in press). Evidently, whether or not a dye reacts with elastin and/or collagens is determined by factors other than its charge.

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As already mentioned, aldehyde-fuchsin showed little or no reaction with renal basement membranes in sections oxidized with periodic acid. These observations confirm earlier pictures by Scott and Clayton (1953). Binding of aldehyde-fuchsin by various tissue structures in oxidized sections probably occurs mainly at sites other than aldehyde groups. In the periodic acid-sodium bisulfite procedure, sulfonic acid groups are introduced according to the equation R-CHO+HSO3- ~ R-CHOH-SO3(Hfickel, 1961). In such pretreated sections aldehyde-fuchsin colors basement membranes intensely, i.e. the dye can approach these sites without difficulty. It seems therefore permissible to assume that failure of aldehyde-fuchsin to react with aldehyde groups in basement membranes is not due to steric hindrance. The interaction of aldehyde-fuchsin with elastin and pseudo-elastic collagen is not understood. Aldehyde-fuchsin in absolute ethanol leaves elastin unstained, but Schwalbe's sheaths and pseudo-elastic fibers are colored; in this respect it resembles other so-called elastica stains (Puchtler et al., 1976). However, in previous investigations of resorcin-fuchsin (Puchtler and Sweat, 1963, 1964b), comparative studies of aldehyde-fuchsin showed fundamental differences in the reaction mechanisms of these two stains. The increased binding of aldehydefuchsin by deaminated sections is probably due to the positive charge of the dye. Acetylation had little effect on staining patterns of aldehyde-fuchsin, but benzoylation induced dye binding by collagen, reticulum fibers and various other structures. Since acetylation and benzoylation both block - NH 2 and - OH groups, the effects of benzoylation cannot be ascribed to the inactivation of these groups. Previous studies indicated interaction of certain dyes with aromatic rings introduced into tissues by benzoylation (Puchtler et al., in press). Perhaps aldehyde-fuchsin can react via similar mechanisms. Recently, Mowry (1978) suggested that aldehyde-fuchsin colors cystine-containing peptides and proteins, such as elastic fibers. This hypothesis seemed to offer an explanation of the intense staining of pseudo-elastic collagen because these fibers apparently belong to the type III collagen group (Puchtler et al., 1976; B6ck, 1977). Type III collagen contains cystine (Chung and Miller, 1974; Gallop and Paz, 1975); the very poor solubility of type III collagen has been ascribed to these cross-links (Gross, 1974; Trelstad, 1974). However, cystine has been demonstrated also in type IV collagens (Clark and Kefalides, 1974; Trelstad, 1974). In histochemical studies B6ck (1978) visualized cystine in renal basement membranes. Since aldehyde-fuchsin does not color basement membranes, cystine apparently does not play a role in the binding of this dye under the conditions tested. During the last decades, dye binding by interactions other than salt-type linkages, hydrogen and covalent bonds has received much attention in physicochemical studies of textile dyeing (McGregor, 1974; Rattee and Breuer, 1974). However, since polycationic dyes have not achieved much commercial success (Baer, 1971), information concerning their reactions with various substrates are scarce. Furthermore, in contrast to other dyes, aldehyde-fuchsin is not a sharply defined chemical entity, but a mixture of poorly known compounds. It was therefore not possible to apply recent developments in dyeing theory to the aldehyde-fuchsin stain.

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Acknowledgement. We thank Mrs. Karen S. McBride for the photographic work. References Baer, D.A. : Cationic dyes for synthetic fibers. In: The Chemistry of Synthetic Dyes. K. Venkataraman (ed.), Vol. IV, p. 161-210. New York and London: Academic Press 1971 Bangle, R. : Gomori's paraldehyde-fuchsin stain. I. Physico-chemical and staining properties of the dye. J. Histochem. Cytochem. 2, 291-299 (1954) B6ck, P.: Staining of elastin and pseudo-elastica ("elastic fiber microfibrils", type III and type IV collagen) with paraldehyde-fuchsin. Mikroskopie 33, 332-341 (1977) B6ck, P. : Histochemical demonstration of type IV collagen in the renal glomerulus. Histochemistry 55, 269-270 (1978) Bolley, P.A., Kopp, E.: Die Theerfarbstoffe. Erster Theil, 1867-1874. Braunschweig: Friedrich Vieweg und Sohn 1897 Chung, E., Miller, E.J.: Collagen polymorphism: characterization of molecules with the chain composition [al(III)]3 in human tissue. Science 183, 1200 120t (1974) Clark, C.C., Kefalides, N.A.: Type IV collagen: a universal component of basement membranes? Devel. Biol. 40, fl-f4 (1974) Denffer, H.v. : Bindungsort und Bindungsmodus von Aldehydfuchsin in den B-Zellen des Inselapparates. Histochemie 36, 97-113 (1973) Denffer, H.v., Heidbrink, V. : Dfinnschichtchromatographische Untersuchung verschiedener Aldehydfuchsine. Acta histochem. (Jena) 48, 62-68 (1974) Fischer, E., Fischer, O.: Uber Triphenylmethan und Rosanilin. Justus Liebigs Ann. Chem. 194, 242-303 (1878) Gallop, P.M., Paz, M.A. : Posttranslational protein modifications, with special attention to collagen and elastin. Physiol. Rev. 55, 418-487 (1975) Gattermann, L., Wichmann, G.: Uber Aldehydblau. Ber. Dtsch. chem. Ges. 22, 227-236 (I889) Goldstein, D.J.: Ionic and non-ionic bonds in staining, with special reference to the action of urea and sodium chloride on the staining of elastic fibers and glycogen. Quart. J. Microsc. Sci. 103, 477-492 (1962) Gomori, G. : Aldehyde-fuchsin: a new stain for elastic tissue. Amer. J. Clin. Pathol. 20, 665-666 (1950) Griffiths, J. : Colour and Constitution of Organic Molecules. London, New York: Academic Press 1976 Gross, J.: Collagen biology: structure, degradation and disease. The Harvey Lectures 68, 331 432 (1974) Gustavson, K.H. : The Chemistry of Tanning Processes. New York: Academic Press 1956 Hadley, E.H. : Aldehydes. In : Encyclopedia of Chemistry, 3rd ed. C.A. Hampel, G.G. Hawley (eds.), p. 41-42. New York: Van Nostrand 1975 Halmi, N.S., Davies, J. : Comparison of aldehyde-fuchsin staining, metachromasia and periodic acid-Schiff reactivity of various tissues. J. Histochem. Cytochem. 1, 447-459 (1953) Harms, H.: Handbuch der Farbstoffe ffir die Mikroskopie. Teil II, 3. Lieferung. Kamp-Lintfort: Staufen Verlag 1958 Hfickel, W. : Organische Chemic. 2. Aufl. Leipzig: Akademische Verlagsgesellschaft 1941 Hfickel, W. : Theoretische Grundlagen der orgauischen Chemic, I. Band, 9. Aufl. Leipzig: Akademische Verlagsgesellschaft Geest & Portig 1961 Magnus: Aldehydgriin. In: Enzyklop/idie der Mikroskopischen Technik. P. Ehrlich, R. Krause, M. Moose, H. Rosin and K. Weigert (eds.), Bd. I, p. 14. Berlin, Wien: Urban & Schwarzenberg 1910 McGregor, R. : Diffusion and Sorption in Fibres and Films. Vol. I. London, New York: Academic Press 1974 McOmie, J.F.W.: Protective Groups in Organic Chemistry. London, New York: Plenum Press 1973 Meloan, S.N., Puchtler, H. : A re-investigation of early elastica stains Anat. Rec. (in press) Meyer, R., Gnehm, R.: Die Theerfarbstoffe. Dritter Theil (1895 1897). Braunschweig: Friedrich Vieweg und Sohn 1897

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Miller, W.v., P16chl, J.: fJber Aldehydgrfin. Ber. Dtsch. chem. Ges. 24, 1700 1715 (1891) Mowry, R.W.: Aldehyde fuchsin staining, direct or after oxidation: problems and remedies, with special reference to human pancreatic B cells, pituitaries, and elastic fibers. Stain Technoi. 53, 141 154 (1978) Nietzki, R. : Chemie der organischen Farbstoffe. 5. Aufl. Berlin: Springer 1906 Ortman, R., Forbes, W.F., Balasurbramanian, A. : Concerning the staining properties of aldehyde basic fuchsin. J. Histochem. Cytochem. 14, 104-111 (1966) Pearse, A.G.E.: Histochemistry, Theoretical and Applied, 3rd ed., vol. 1. Boston: Little Brown & Co. 1968 Prud'homme, M. : Nouvelle synth6se de la parafuchsine et de ses d6riv+s mono-, di-, tri- et t6traalcoyl6s. Bull. Soc. Chem. 15, 142-146 (1896) Puchtler, H., Sweat, F., Bates, R., Brown, J.H.: On the mechanism of resorcin-fuchsin staining. J. Histochem. Cytochem. 9, 553-559 (1961 Puchtler, H., Sweat, F. : Influence of various pretreatments on the staining properties of connective tissue fibers. Ann. d'Histochim. 8, suppl, I, 189-198 (1963) Puchtler, H., Sweat, F. : A selective stain for renal basement membranes. Stain Technol. 39, 163-166 (1964a) Puchtler, H., Sweat, F.: Histochemical specifity of staining methods for connective tissue fibers: Resorcin-fuchsin and Van Gieson's picro-fuchsin. Histochemie 4, 24 34 (1964b) Puchtler, H., Meloan, S.N., Brewton, B.R.: On the history of basic fuchsin and aldehyde-Schiff reactions from 1862 to 1935. Histochemistry 41, 185-194 (1975) Puchtler, H., Meloan, S.N., Pollard, G.R. : Light microscopic distinction between elastin, pseudoelastica (type III collagen?) and interstitial collagen. Histochemistry 49, 1-14 (1976) Puchtler, H., Waldrop, F.S., Meloan, S.N. : Effects of acetylation and benzoylation on dye binding: Investigation of molecular alterations in models. Histochemistry 58, 65-70 Rath, H. : Lehrbuch der Textilchemie. 3. Aufl. Berlin, Heidelberg, New York: Springer 1972 Rattee, I.D., Breuer, M.M.: The Physical Chemistry of Dye Adsorption. London, New York: Academic Press 1974 Schiff, H.: Note sur Faction des aldehydes sur la rosaniline. C.R. Acad. Sci. (Paris) 61, 45-75 (1865) Schiff, H.: Eine neue Reihe organischer Diamine. Justus Liebigs Ann. Chem. 140, 92-137 (1866) Schubert, F. : Lehrbuch der technischen Chemie. 2. Aufl. Erlangen: Ferdinand Enke 1866 Scott, H.R., Clayton, B.P.: A comparison of the staining affinities of aldehyde-fuchsin and the Schiff reagent. J. Histochem. Cytochem. 1, 336 346 (1953) Sumner, B.E.H.: A histochemical study of aldehyde fuchsin staining. J. Royal Microsc. Soc. 84, 329-338 (1965) Trelstad, R.L. : Vertebrate collagen heterogeneity. Devel. Biol. 38, f13-f16 (1974) Venkataraman, K.: The Chemistry of Synthetic Dyes. Vol. i. New York: Academic Press 1952 Wagner, E.C.: A rationalization of acid-induced reactions of methylene-bis-amines, methyleneamines, and of formaldehyde and amines. J. Org. Chem. 19, 1862-1881 (1954) Walker, J.F. : Formaldehyde. 3rd ed. New York: Reinhold 1964 Younker, T.D., Waldrop, F.S., Puchtler, H.: Dye binding by collagens and elastin: Effects of dye configurations and solvents. J. South Carolina Med. Ass. 74, 59 (1978)

Received December 6, 1978