fluorescence of trypan blue in frozen-dried embryos of the rat

Histochemistry 54, 177- 189 (1977) Histochemistry �9 by Springer-Verlag 1977

Fluorescence of Trypan Blue in Frozen-Dried Embryos of the Rat*

Howard W. Davis and Ronald W. Sauter Department of Anatomy, School of Medicine, University of Oregon Health Sciences Center, Portland, Oregon 97201, USA

Summary. Freeze-drying and fluorescence microscopy techniques were com- bined to create a sensitive method for the visualization of the teratogenic dye, Trypan blue, in both protein-bound and free forms. In the development and initial application of this method, visceral yolk sacs of several gestational ages as well as normal appearing, 12-day embryos obtained from dye-injected rats were utilized. Observations on paraffinized sections of the yolk sac placentae demonstrated that only the protein-bound form of the dye exists in the yolk sac cavity whereas both forms of the dye exist in supranuclear regions of cells of the visceral endoderm. Paraffin sections of the normal appearing, 12-day embryos displayed the protein-bound form of dye within lumina of mid- and hind-gut, and both forms of dye in the primitive mucosa of mid- and hind-gut. The advantages of the method are derived not only from the use of fluorescence microscopy but also from the avoidance of solvents that are employed in more routine microtechniques.

Introduction

Since the 1948 report by Gillman, Gilbert, Gillman and Spence of the teratogenic action of Trypan blue on the rat embryo, the inability of investigators to detect this disazo dye within the rodent embryo or fetus has discouraged the formula- tion of studies based on the hypothesis of a direct teratogenic action on embryonic tissues. Davis and Gunberg (1968), however, presented evidence of Trypan blue in the gut of rat embryos after dye injection into the maternal animal on the 9th day of pregnancy. That report utilized two routine procedures: low power examination (15 x magnification) of unstained whole mounts of 75 experimental embryos of 11, 12 and 13 days of gestation, and light-microscopy

* Supported in part by the Oregon Heart Association and by the Medical Research Foundation of Oregon, grant 7513

178 H.W. Davis and R.W. Sauter

of stained, paraffin sections of experimental embryos of 12 days of gestation. Each of the 75 unstained whole mounts displayed blue color in mid- and hind- gut. In these whole mounts, the pattern of dye distribution was constant for the embryos within any one of the three age groups. The stained, paraffin sections of 12-day embryos revealed faint blue vesicles of Trypan blue in endoder- real cells of the gut, cloaca and allantois, and in adjacent mesenchymal cells. Those areas also contained basophilic granules, which were more prevalent in tissues from the experimental series than in tissues from the control series. In stained sections of embryos in the experimental series, sites of incipient malformation (e.g., optic and otic primordia) displayed blue vesicles and basophilic granules, but neither kind of inclusion could be unequivocally identified as Trypan blue.

Any search for intra-embryonic Trypan blue must consider the two kinds of problems encountered in the previous study. First, routine methods of preparation for light-microscopy expose embryonic tissues to a variety of solvents during fixation, dehydration, clearing and paraffin infiltration. These solvents solubilize, decolorize or cause dislocation of some portion of the intra-embryonic dye (see Discussion). Second, the staining step not only exposes embryonic tissues to further solvent action but also adds color which can impair the identification of the blue teratogen.

This report presents considerations that led to the development of an improved method for the detection and characterization of intra-embryonic dye. In this method, the technique of freeze-drying eliminated the need for the majority of the solvents ordinarily used in the preparation of tissues for light-microscopy. In addition, a phenomenon of fluorescence permitted protein- bound Trypan blue to be distinguished from dye not bound to protein. Steinwall and Klatzo (1966) observed that a mixture of Trypan blue and bovine albumin emitted a red fluorescence under ultraviolet light. Hamberger and Hamberger (1966) observed that neither Trypan blue nor serum albumin produced any fluorescence when examined separately. In the present study, the nonspecific, background fluorescence (green) of embryonic cells served as an adequate substitute for counterstaining with basic or acid dyes.

The development and initial application of this method utilized two kinds of embryonic tissues: visceral yolk sacs of several gestational ages and normal appearing, 12-day embryos obtained from Trypan blue injected pregnant rats. Visceral yolk sacs were used to study the effects of modification of several phases of the method because cells of that placental envelope are known to phagocytize Trypan blue (Waddington and Carter, 1953; Hamburgh, 1954; Wilson, 1955). Furthermore, dye probably enters the phagocytic cells of visceral yolk sacs in protein-bound form (Lloyd and Beck, 1969), so these cells might be expected to display fluorescent dye inclusions. The method was applied to 12-day embryos because the unstained whole mounts of the previous study (Davis and Gunberg, 1968) demonstrated that gut regions of 11- to 13-day embryos of the experimental series contained high concentrations of intra-embryonic dye. Twelve-day embryos that showed external malformations or irregular heart functions were excluded from this study, since a small percentage of such embryos die in later stages of gestation (Beck and Lloyd, 1963).

Fluorescence of Trypan Blue in Embryos

Material and Methods

179

Model Experiments on the Fluorescence of Trypan Blue

The Trypan blue solutions used in this study were freshly prepared from a crystalline dye sample previously utilized in the production of a variety of malformations in rat embryos (Davis and Gunberg, 1968). To ensure that this sample of Trypan blue (Lot no. 2062P, C. 1. no. 23850; National Aniline Division, Allied Chemical Corporation) did not contain fluorescent impurities, the following experiment was performed. Trypan blue was dissolved in distilled water in concentrations of 15, 0.15 and 0.0015 mg/ml. One gl spots of each of these three solutions were air dried on glass slides for examination by ultraviolet microscopy.

Experiments on Visceral Yolk Sacs

To test the effects of modifications in technique on a tissue widely reported to phagocytize Trypan blue, visceral yolk sacs were obtained from 10- and 14-day embryos, both experimental and control. Preliminary investigations prompted the selection of placental tissues of these ages (see Results). The yolk sacs were processed as described below for 12-day embryos, with the following exception. In the procurement of 10-day yolk sac tissues, the entire uterus was bisected on either side of the implant and then snap-frozen as described. Visceral yolk sacs also were used to test the effects of two kinds of vapor fixation on dye-containing, desiccated tissues. Prior to vacuum infiltration with paraffin, frozen-dried yolk sacs were post-fixed either for I h at 37 ~ C in a closed chamber containing crystals of osmium tetroxide' or for 2 h at 51 ~ C in a closed chamber containing parafor- maldehyde.

Definitive Technique and its Application to 12-Day Embryos

Rats of the Sprague-Dawley strain were supplied with pelleted rat food and water ad libitum. Females were mated with males of their own strain during a two-hour period. The age of an embryo, expressed in days, was computed by dividing by 24 the number of hours intervening between the midpoint of this two-hour mating period and the initiation of the maternal laparotomy. At eight days plus four hours of gestation the pregnant rats were injected intraperitoneally with a 1.5% aqueous solution of Trypan blue at a dosage level of 75 mg/kg body weight. At this time of injection, this dosage level is 17% less than our "opt imal teratogenic dose" (unpublished data). Maternal animals in the control group received no injections. Ninety two hours later, the pregnant rats were anesthetized with Nembutal . Each 12-day embryo was dissected away from the envelope of uterine musculature and placed on a copper disk. After the remaining uterine tissues and those extra-embryonic membranes peripheral to the amnion were removed and discarded, the amuion was pulled apart and the embryo was briefly examined under a dissecting microscope. Embryos that displayed external malformat ion or abnormal heart function were excluded from this study. The tissue-laden disk was submerged in isopentane cooled to about - 1 6 0 ~ with liquid nitrogen (Pearse, Appendix 3, 1968), and transferred on dry ice to the cold platen of an Edwards- Pearse model III freeze-dryer. The desiccated, unfixed embryos were vacuum embedded in paraffin and serially sectioned at 10 ~. Sections were mounted on clean, dry glass slides, without cover slips, for examination with a Zeiss Universal microscope fitted with a type II (F1) epi-illumination device and Planachromat lenses. Magnifications on film of 315 required the use of a special 63 • objective (0.90 N.A.) which was corrected for use with unslipped specimens. An HBO 200 (Osram) burner with a Zeiss BG 12 excitation filter was used as an ultraviolet light source, and a Zeiss " 5 0 " filter as a barrier filter. Daylight Kodachrome (Kodak; ASA 64) was used for fluorescence and tungsten-light photomicrography; Photomicrography Color Film ~2483 (Kodak; ASA 16) was also used during the latter mode of illumination. A substage filter was used to improve color balance in tungsten-light photography.

180

Results

Model Experiments on the Fluorescence of Trypan Blue

None of the 1 gl, air dried spots of aqueous solutions of Trypan blue exhibited fluorescence under ultraviolet illumination.

H.W. Davis and R.W. Sauter

Experiments on Visceral Yolk Sacs

A very brief, histological description of the inner (i.e., visceral) wall of the rat yolk sac is included here for convenience in the interpretation of the results. The visceral wall of the rat yolk sac has two epithelial surfaces. An absorptive, simple columnar epithelium (i.e., visceral endoderm) borders the yolk sac ca,)ity, and a mesothelium delimits the exocoelom. Between these two epithelia lies a layer of mesenchyme in which vitelline vessels are found. For detailed cytological and histological descriptions of the yolk sac placenta, consult Padykula and Richardson (1963).

Preliminary investigations on visceral yolk sacs prompted the selection of 10-day tissues for more extensive study, because as compared with 12-day tissues, the cells of the younger placentae contained larger, more intense areas of red fluorescence. The fate of these areas of red fluorescence was studied in 14-day visceral yolk sacs.

Tungsten-light microscopy of paraffin sections of unstained, 10-day visceral yolk sacs of the experimental series revealed evidence of dye in vacuoles within cells of the visceral endoderm (Fig. 1). Faint blue vacuoles were located close to the apical ends of these cells. Dark blue vacuoles were situated in deeper regions of the supranuclear areas. With ultraviolet illumination of these same fields of view (Fig. 2), the vacuoles that were faint blue in tungsten light now exhibited a red fluorescence, which signified the presence of protein-bound dye. The vacuoles that were dark blue in tungsten light now appeared as black inclusions, which signified that the dye was no longer bound to protein. In addition, ultraviolet light revealed a large number of fluorescent, intracellular vacuoles of dye that could not be detected with the tungsten-light mode of illumination. Fluorescent deposits of dye also could be detected in yolk sac cavities and in maternal blood spaces. In general, ultraviolet illumination revealed much greater amounts of dye in the visceral yolk sac than did tungsten illumination.

Microscopic examination of paraffin sections of unstained, 14-day visceral yolk sacs of the experimental series revealed a different pattern of dye deposition in the visceral endoderm. Using tungsten illumination (Fig. 3), deposits of highly concentrated dye were seen in the supranuclear regions of these cells. Nearly all of these dye deposits were dark blue and irregular in outline; faint blue vacuoles were seen rarely. With ultraviolet illumination of these same fields of view (Fig. 4), the dark blue deposits seen with tungsten illumination now appeared as black inclusions. Masses of small, red, fluorescent vacuoles of

Fluorescence of Trypan Blue in Embryos 18i

protein-bound dye were situated between the non-fluorescent deposits of dye and the apical surfaces of these endodermal cells. Red fluorescence coated the microvillous surfaces of most of these cells.

In unstained visceral yolk sacs of the experimental series of both 10- and 14-day embryos, no dye was detected in the vitelline vessels or in layers closer to the exocoelom than the visceral endoderm, regardless of the mode of illumination. Furthermore, in no instance did the comparable, control tissues display images that indicated the presence of either form of the dye.

Preliminary experiments provided the reasons for the utilization of dry mount preparations of visceral yolk sacs in the present study. Sections of tissues embedded in paraffin usually are floated on warm water onto a glass slide previously coated with a thin layer of albumin-glycerol, a section adhesive. Because both the warm water, used to expand the paraffin, and the glycerol component of the adhesive caused considerable solubilization and translocation of dye inclusions, these steps were avoided. Furthermore, a deparaffinizing step was excluded from the present method, because the application of toluene or xylene to paraffin sections eliminated much of the red fluorescence. Mounting media such as immersion oil, paraffin oil, clove oil, methyl salicylate and pyridine were applied to paraffin sections prior to application of cover slips. Of these substances, immersion oil and paraffin oil were the least destructive of red fluorescence, but after a period of several days the red fluorescence was barely detectable. In consideration of these findings, microscopic observations were made on dry paraffin sections, using neither floating media, section adhesives nor cover slips.

The present technique of tissue preparation also was modified by the results of experiments that tested the effects of vapor fixation on desiccated visceral yolk sacs of various ages. The osmium tetroxide vapor procedure greatly reduced the intensity of both autofluorescence and red fluorescence, as compared with unfixed tissues. The exposure of frozen-dried tissues to formaldehyde vapor was much less harmful; as compared with unfixed tissues, only a slight reduction occurred in intensity of red fluorescence, and the background fluorescence was not altered. Therefore, some of the embryos used in later phases of this study were post-fixed in formaldehyde vapor. Such vapor fixation permitted the routine staining, in solvents, of alternate paraffin sections in order to improve orientation. Figures 3 and 4 illustrate visceral yolk sacs that were post-fixed in formaldehyde vapor and not exposed to acid or basic counterstains. Although adjacent paraffin sections stained with hematoxylin and eosin (not illustrated) displayed excellent morphology, the staining procedures either removed, or reduced the size of, many of the deposits of dye. In both fixed and unfixed tissues that were subjected to lengthy examination with ultraviolet illumination, the red fluorescence resisted fading much better than did the green, background fluorescence.

Experiments on 12-Day Embryos

Microscopic examinations of unstained, paraffin sections of 12-day embryos of the experimental series revealed unequivocal evidence of dye. Under ultravio-

182 H.W. Davis and R.W. Sourer

let i l luminat ion, the most intense areas of red fluorescence were seen within mid- and h ind-gut lumina (Figs. 5 and 6). The largest areas of such fluorescence were located within the tail gut opposite the al lantoic divert iculum. The red fluorescence extended vent rad for a short distance into the al lantoic stalk. F r o m the allantoic diver t iculum anteriorly to a level just caudal to the liver, gut lumina were filled with red fluorescence of lesser intensity. In sections of two embryos f rom which the visceral yolk sacs had not been removed, the intensi ty of the red fluorescence within mid- and h ind-gut lumina always exceeded that within the endodermal cells of the visceral yolk sacs (Fig. 6). Of the total n u m b e r of sections of mid- and hind-gut , only a few gave no evidence of red fluorescence within gut lumina (Figs, 7 and 8). Tungs ten i l lumina t ion did no t reveal evidence of the dye conta ined within the gut lumina in these 10 la sections.

Microscopic examinat ions of unstained, paraffin sections of 12-day embryos of the experimental series also revealed unequivocal evidence of dye within the mucosa of mid~ and hind-gut , and much lesser amoun t s of dye within areas peripheral to the developing mucosa, Tungs ten i l lumina t ion was useful in the initial detection of most of these dye deposits (Figs. 5 a nd 7); ul traviolet i l lumina- t ion of these same fields of view allowed these major deposits, which were

Fig. 1, Yolk sac from a 10-day embryo, experimental series; tungsten illumination. Compare with Figure 2 which represents the identical field of view under ultraviolet illumination. In this photomicrograph of a paraffin section of unfixed, unstained tissue, note the various shades of blue color displayed by vacuoles within endodermal cells of the visceral wall of the yolk sac. Two of these vacuoles (below the asterisks) contain high concentrations of Trypan blue. Other vacuoles, situated closer to the yolk sac cavity (arrows), are difficult to detect because they contain very low concentrations of the dye. Kodachrome film x 315

Fig. 2. Yolk sac from a 10-day embryo, experimental series; ultraviolet illumination. Compare with Figure 1 which represents the identical field of view under tungsten illumination. Each of the faintly colored, endodermal cell vacuoles referred to in Figure 1 now displays red fluorescence, which is characteristic of protein-bound Trypan blue. In addition, red fluorescence demarcates endodermal cell vacuoles that cannot be seen under tungsten illumination. Red deposits of dye also can be seen in the yolk sac cavity (arrows) and in the maternal blood spaces (lower half of figure). The two vacuoles of highly concentrated Trypan blue seen under tungsten illumination (Fig. 1) now appear black, which indicates that the dye is not bound to protein. The yellow-green flare at the upper right of the photomicrograph masks tissue components that separate the large, green embryonic blood cells in the vitelline vessel from the exocoelom (dark region at upper left of figure)~ Kodachrome film x 315

Fig. 3. Visceral walI ofa yolk sac from a 14-day embryo, experimental series; tungsten iUumination. Compare with Figure 4 which represents the identical field of view under ultraviolet illumination. In this photomicrograph of a paraffin section of formaldehyde vapor-fixed, unstained tissue, note the supranuclear deposits of highly concentrated Trypan blue in cells of the endodermal epithelmm. Diffuse outlines of embryonic blood cells can be seen within the large vitelline vessel situated close to the exoeoelom (left margin of figure), Maternal blood cells (e.g., upper right of figure) can be seen adjacent to the apical ends of cells of the endodermal epithelium; this dislocation is an artifact produced during dissection of the visceral yolk sac from surrounding tissues. Photomi- crography Color Film #2483 x 315

Fluorescence of Trypan Blue in Embryos 183

Fig. 4. Visceral wall of a yolk sac from a 14-day embryo, experimental series ; ultraviolet illumination. Compare with Figure 3 which represents the identical field of view under tungsten illumination. Note the masses of red vacuoles of protein-bouild Trypan blue situated in the apices of the simple columnar cells of the visceral endoderm. The microvillous surfaces of these cells also display red fluorescence. The blue deposits of highly concentrated Trypan blue seen under tungsten illumination (Fig. 3) appear black under ultraviolet illumination. During direct examination of this field of view, these black, supranuclear deposits of protein-free dye could be distinguished from the dark, oval-shaped nuclei. As a result of focussing on the red images, the configurations of the deposits of protein-free dye seen in this photomicrograph vary slightly from those seen under tungsten illumination (Fig. 3). Ultraviolet illumination provides more distinct images of unstained cells tha~ does tungsten illumination. Kodachrome film x 315


Fig. 5. Cross section of tail region from a 12-day embryo, experimental series; ultraviolet illumination of unfixed, unstained tissue. Green autofluorescence clearly demarcates the neural tube, notochord and hind-gut. Observe the bright red protein-bound Trypan blue within the lumen of the gut. Direct observation of this section at higher magnification revealed a red deposit of dye (barely visible at lower arrow) in the mucosa of the primitive gut. Under tungsten illumination, direct observation of this section at higher magnification revealed two deposits of blue dye (black dots, barely visible at upper arrow) in the mucosa, but no evidence of the dye seen here within the lumen of the gut. Kodachrome film • 80

Fig, 6. Sagittal section of mid-gut region from a 12-day embryo, experimental series; ultraviolet illumination of unfixed, unstained tissue. Observe the bright red protein-bound Trypan blue within the lumen of the gut. Under tungsten illnmination, direct observation of this section revealed no evidence of the dye within the lumen of the gut. Note the greater intensity of the red fluorescence


devoid of both red fluorescence and green autofluorescence, to be localized to the primitive mucosa (Fig. 8). Ultraviolet illumination also permitted the identification of a few, distinctly red, fluorescent deposits of protein-bound dye within the mucosa (Figs. 5 and 8). The majority of these fluorescent deposits d~d not dispiay blue color uader tungstert illuminatio~ (compare Fig. 7 with Fig. 8). Smatl deposits of either or both forms of lhe dye were occasionally seen within areas peripheral to the developing mucosa.

Using both modes of illumination, microscopic examinations of unstained, paraffin sections of 12-day embryos of the control series revealed no images that resembled either form of the dye.

Discussion

Some investigators have recognized the deleterious effects on intravital dyes of many of the solvents commonly employed in the preparation of tissues for microscopy. Thilander (1964) demonstrated that Bouin's fluid, the "most suitable" of nine fixatives tested on tissues containing Trypan blue, did show a "slight decolourizing action." Both Pertussa (1966) and Thilander (1964) noted the solubilizing and decolorizing effects on vital dyes of the ethyl alcohol solutions routinely employed in dehydration. Williams and Frant2 (1948) used acetone dehydration to avoid dye solubilization, and Williams (1948) used acetone both as a fixative and as a stain solvent. In an unpublished experiment, however, we used acetone to partially dehydrate Bouin's-fixed embryos prior to final dehydration in a critical-point drying apparatus (Freon 113, Freon 13 sequence). This dehydration procedure did not prevent dye solubiliTation nearly as well as that of freeze-drying. Williams and Frantz (1948) noted that vital dyes within damaged, adult epithelial cells either were obscured by an added stain or were extracted during t le staining procedure.

within the lumen of the gut as compared with that in cells of the endodermal epithelium of the visceral yolk sac (5). Protein-free dye can be identified as small, black aggregates in the visceral yolk sac. Kodachrome film • 80

Fig. 7. Sagittal section of hind-gut region from a 12-day embryo, experimental series; tungsten illumination of unfixed, unstained tissue. Compare with figure 8 which represents the identical field of view under ultraviolet illumination. Note the numerous deposits of concentrated Trypan blue situated in tissues that border the barely discernible, oblique sections of Inmina (asterisks) of the gut. Kodachrome film x 315

Fig. 8. Sagittal section of hind-gut region from a 12-day embryo, experimental series; ultraviolet illumination of unfixed, unstained tissue. Compare with Figure 7 which represents the identical field of view under tungsten illumination. The deposits of dye that were blue under tungsten illumination (Fig. 7) now appear black. The gut mucosa also contains dye in the red~ fluorescent form In this particular section, the lumina of the gut display no evidence of dye (compare with Figure 5), Kodachrome film x 315


The present study provided additional cautions against the use of solvents on dye-containing tissues. The experiments on visceral yolk sacs indicated that Trypan blue was solubilized by water flotation of sections, section adhesives, staining procedures and mounting media. In 12-day embryos, freeze-drying permitted the identification of more blue dye than did the procedure that utilized solvents for fixation and dehydration (Davis and Gunberg, 1968). It is important to note that, with the exception of paraffin, no solvents were used in the definitive method of tissue preparation for fluorescence microscopy.

The sensitivity of the present technique was due not only to the method of preparation but also to the mode of examination. Fluorescence microscopy is more sensitive than tungsten-light microscopy in the detection of azo dyes (Hamberger and Hamberger, 1966; Telfer and Anderson, 1968; Nairn, 1969; Pearse, p. 1180, 1972). In the present study of both visceral yolk sacs and 12-day embryos, ultraviolet illumination revealed deposits of dye that could not be detected with tungsten illumination. Some indication of the degree of sensitivity was provided by Hamberger and Hamberger (1966), who observed weak but distinct fluorescence in 1 gl, air dried spots of aqueous solutions of serum albumin that contained as little as 5 x 10 -~ mg/ml of Trypan blue.

Three kinds of considerations strengthen the validity of those observations that we have characterized as microscopic images of Trypan blue. First, with respect to tungsten-light examinations, the certainty of identification of the blue dye was enhanced by deletion from the technique of a staining step. In the previous study (Davis and Gunberg, 1968), some of the high-magnification identifications of intraembryonic dye were equivocal because the basic stains, applied for the purpose of orientation, introduced shades of color close ,to that of the native dye. Second, several experiments by other investigators support our interpretations of the red, fluorescent images. In vitro experiments by Steinwall and Klatzo (1966) and Hamberger and Hamberger (1966) demonstrated the red fluorescence of Trypan blue-albumin mixtures. Using aqueous solutions that contained Trypan blue as the only solute, Hamberger and Hamberger (1966) and Kormano (1968) utilized the fluorescent phenomenon of the resultant dye-protein complex to investigate the disruption .of tissue barriers in animals traumatized prior to dye injection. Both the present study and the experiments of Hamberger and Hamberger (1966) showed that neither Trypan blue nor serum albumin fluoresced when examined separately. The bound (i.e., adsorbed) form of Evans blue, a teratogen and an isomer of Trypan blue, also displays red fluorescence (Steinwall and Klatzo, 1966; Pearse, p. 1422, 1972). Trypan blue and Evans blue are aminonaphthalene sulfonic acids, compotmds~, that fluoresce when adsorption to protein distorts the dye molecule into a planar configuration (Udenfriend, 1962). Lastly, in support of the validity of our interpretations, indications of dye seen with tungsten light were substantiated by Observations with ultraviolet light, and vice versa.

For the purpose of orientation during the microscopic observations, the green, background autofluorescence served as an adequate substitute for counterstaining. During photography, however, lengthy exposure of tissues to ultraviolet light caused a fairly rapid fading of portions of the autofluorescent background. Hamberger and Hamberger (1966) also noted the durability of the red fluores-


of the somes Beck, 1968) cells.

A

cence, as compared with that of the autofluorescence. Therefore, in later phases of this study, we stained alternate sections in order to improve orientation. The preparation of stained, alternate sections required that desiccated tissues be post-fixed, but not by the use of solvents. Fixation by vapors, a useful technique for almost all histochemical methods (Pearse, 1968), is especially valuable in its present application to desiccated tissues that contain Trypan blue in non-sequestered, relatively unprotected sites (e.g., gut lumina). The particular formaldehyde vapor procedure used in this study caused a very slight diminution in intensity of the red fluorescence seen in dry-mounted sections. The degree of that reduction, however, was not sufficient to disallow such future use of formaldehyde vapor.

Several investigators have contributed to an understanding of the processes that culminate in dye uptake by the visceral yolk sac. After parenteral administra- tion, Trypan blue binds to the albumin fraction of plasma proteins (Rawson, 1943 ; Lloyd and Beck, 1968). At the dosage levels ordinarily employed, probably none of the dye exists in free form in plasma (Lloyd et al., 1968). In the rat, this dye-protein complex confronts no barrier in its passage from maternal blood to yolk sac cavity (Jollie, 1968). Presumably, then, the endodermal cells

visceral yolk sac endocytize the dye, which accumulates in heterophago- ; subsequently, the dye is concentrated in heterolysosomes (Lloyd and 1968; Lloyd et al., 1968). Electron microscope observations (Lloyd et al., provide no evidence concerning exocytosis of dye from the endodermal

summary of our observations allows some interesting comparisons with those results obtained from more definitive studies. Our observations of 10- and 14-day visceral yolk sacs of the experimental series suggest the following pattern of dye uptake by the endodermal cells. Protein-bound dye, absorbed from the yolk sac cavity, appears within intracellular vacuoles. As the dye-laden vacuoles mo;ee from an apical position toward deeper regions of the supranuclear areas of these absorptive ceils, the dye is gradually deprived of its association with protein, probably through the action of lysosomal proteases. In older placentae, many of the dye deposits situated closely adjacent to the apical ends of nuclei have coalesced into granular, protein-free masses. Our observations demonstrate not only that the dye exists in bound (i.e., fluorescent) form in maternal blood and yolk sac cavity but also that little or no protein exists in association with the most concentrated deposits of dye within endodermal cells. Although the present report characterizes one form of Trypan blue as "protein-bound," it is possible that substances other than protein adsorb Trypan blue and thus impart fluorescence to a dye-containing macromolecule. The in vitro experiments of Hamberger and Hamberger (1966) demonstrated the red fluorescence in mixtures of Trypan blue and sucrose. Moreover, it has been suggested that lipid, as well as protein, can be absorbed by pinocytosis by neonatal absorptive cells of the ileum (see references in Cornell and Padykula, 1969), and it is not known that lipid cannot bind with, and thus impart fluorescence to, Trypan blue.

The 12-day embryos of the experimental series displayed extensive evidence of Trypan blue within the lumina and primitive mucosa of mid- and hind-gut;


the mesenchyme adjacent to these areas displayed much less evidence of dye. These luminal areas contained dye wholly in protein-bound form, whereas the mucosal and surrounding, mesenchymal areas showed dye in both bound and free forms. The observations suggested that protein-bound dye was being transferred from the lumina into the mucosa, and that the extent of transfer varied among the different, gross levels of mid- and hind-gut.

The gross pattern of dye distribution within mid- and hind-gut lumina coin- cides with that seen by low power examination of unstained whole mounts of 12-day embryos in the previous study (Davis and Gunberg, 1968). Although neither study attempted to determine the significance of such dye localization, it is presumed that Trypan blue is admitted to the gut lumen from the yolk sac cavity at a time prior to closure of this route of communication. Indeed, as Hamburgh et al. (1975) have pointed out, "'cells of the head process, early notochord and possibly the primitive streak are exposed to fluids in the yolk sac cavity prior to the complete envelopment of the embryo by the yolk sac."

The reported failure of Trypan blue to penetrate embryos has been ascribed not only to placental barriers but also to properties of the intraembryonic tissues themselves. This latter rationale is based on the failure to detect dye within tissues of amphibians (Waddington and Perry, 1956; Greenhouse and Hamburgh, 1968), which lack a yolk sac envelope, and of chick and rat embryos cultivated in vitro (Mulherkar, 1960; Turbow, 1966). A concept of dye action on the surfaces of embryonic cells would not conflict with the results of these kinds of experiments. As an alternate hypothesis to surface action, it is also conceivable that dye can enter cells of the rat embryo, by whatever route, in small but effective quantities, yet not be detected with routine methods. In future studies, the sensitivity of the present method offers the possibility that intraembryonic dye can be detected in embryos younger than twelve days of gestation, perhaps in anlagen of organ systems known to display high fre- quencies of malformation.

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Received June 16, 1977

fluorescence of trypan blue in frozen-dried embryos of the rat

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