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

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<ul><li><p>Histochemistry 54, 177- 189 (1977) Histochemistry 9 by Springer-Verlag 1977 </p><p>Fluorescence of Trypan Blue in Frozen-Dried Embryos of the Rat* </p><p>Howard W. Davis and Ronald W. Sauter Department of Anatomy, School of Medicine, University of Oregon Health Sciences Center, Portland, Oregon 97201, USA </p><p>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. </p><p>Introduction </p><p>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 embryo- nic 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 </p><p>* Supported in part by the Oregon Heart Association and by the Medical Research Foundation of Oregon, grant 7513 </p></li><li><p>178 H.W. Davis and R.W. Sauter </p><p>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 baso- philic granules, but neither kind of inclusion could be unequivocally identified as Trypan blue. </p><p>Any search for intra-embryonic Trypan blue must consider the two kinds of problems encountered in the previous study. First, routine methods of prepa- ration 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. </p><p>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 substi- tute for counterstaining with basic or acid dyes. </p><p>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-em- bryonic dye. Twelve-day embryos that showed external malformations or irregu- lar heart functions were excluded from this study, since a small percentage of such embryos die in later stages of gestation (Beck and Lloyd, 1963). </p></li><li><p>Fluorescence of Trypan Blue in Embryos </p><p>Material and Methods </p><p>179 </p><p>Model Experiments on the Fluorescence of Trypan Blue </p><p>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 concentra- tions 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. </p><p>Experiments on Visceral Yolk Sacs </p><p>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. </p><p>Definitive Technique and its Application to 12-Day Embryos </p><p>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 "optimal 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 malformation or abnormal heart function were excluded from this study. The tissue-laden disk was submerged in isopentane cooled to about -160~ 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 "50" 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. </p></li><li><p>180 </p><p>Results </p><p>Model Experiments on the Fluorescence of Trypan Blue </p><p>None of the 1 gl, air dried spots of aqueous solutions of Trypan blue exhibited fluorescence under ultraviolet illumination. </p><p>H.W. Davis and R.W. Sauter </p><p>Experiments on Visceral Yolk Sacs </p><p>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 cytolog- ical and histological descriptions of the yolk sac placenta, consult Padykula and Richardson (1963). </p><p>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. </p><p>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 re- vealed much greater amounts of dye in the visceral yolk sac than did tungsten illumination. </p><p>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 </p></li><li><p>Fluorescence of Trypan Blue in Embryos 18i </p><p>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. </p><p>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 illumina- tion. Furthermore, in no instance did the comparable, control tissues display images that indicated the presence of either form of the dye. </p><p>Preliminary experiments provided the reasons for the utilization of dry mount preparations of visceral yolk sacs in the present study. Sections of tissues embed- ded 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. </p><p>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 r...</p></li></ul>