TISSUE & CELL 1977 9 (2) 347-371 Published by Longman Group Ltd. Printed in Great Britain
M. LOCKE and P. HUIE
STAINING FOR MICROSCOPY
ABSTRACT. Bismuth salts on aldehyde fixed tissue give a highly selective pattern of staining suitable for light and electron microscopy. Structures stained include the nucleolus, ribosomes, inter- and perichromatin granules, the Golgi complex beads and the outer face of the tubule doublets of mouse sperm, certain neurosecretory vesicles believed to contain biogenic amines, some junctions (some central synapses, neuromuscular junctions, tight junctions), specialized membranes such as the post acrosomal dense lamina of mouse sperm and the inner alveolar membrane of Paramecium, and a variety of structures associated with the cytoplasmic face of membranes, such as plasma membrane plaques, cleavage furrows, the leading edge of the spreading acrosome and sperm annuli. Staining is not reduced by nucleases and spot tests show no reaction between nucleic acids and bismuth under conditions similar to those used to stain tissues. However, spot tests do show strong binding of bismuth by basic proteins and by some phosphorylated molecules. It is hypothesized that bismuth reacts with cell components in two ways, distinguishable by their glutaraldehyde sensitivity. For example, staining of the nucleolus and ribosomes is blocked by glutaraldehyde but the inter- and perichromatin granules and the GC beads are unaffected. Spot tests show that basic proteins (histones, protamines, polylysine and polyargenine) and other molecules with free amino groups (5HT, tryptamine, dopamine) bind bismuth strongly, a reaction that is blocked to varying degrees by glutaraldehyde. We presume that most bismuth staining of tissues is due to reaction with amine groups and is glutaraldehyde sensitive and some may be due to guanidine groups which are less sensitive to fixation by glutaraldehyde. Organic phosphates may be the cause of the glutaraldehyde insensitive staining since ATP and some other phosphates bind bismuth in a reaction that is not blocked by glutaraldehyde.
tures such as nucleoli that are resolvable by light microscopy, the bound bismuth can be converted to the brown/black sulphide, or to an intense purple lake with haematoxylin. The staining procedure developed for electron microscopy can also be used to identify components of cell fractions and molecules separated by electrophoresis on cellulose strips. The initial impetus for this work came from the notion that fixed molecules having exposed phosphate groups might be shown up by their capacity to bind bismuth. Earlier workers (Albersheim and Killias, 1963) had suggested that bismuth binds to the exposed phosphate backbone of nucleic acids, an idea perpetuated in current textbooks (Hayat, 1975, p. 301). Our first experiments appeared to confirm this, since we were able to show
Introduction BISMUTH staining on sections gives a relatively non-specific enhancement of contrast (Barrett et al., 1975; Riva, 1974) compared to that obtained by staining the tissue before embedding (Locke and Huie, 1975a). Tissue treated with bismuth salts shows a highly selective pattern of staining with a very fine grain capable of high resolution localization by electron microscopy. It shows components not usually distinguishable from their background and even resolves structures not known to exist after other staining procedures (Locke and Huie, 1975a, 1976a). For struc-~~ The Cell Science Laboratories, Department of Zoology, The University of Western Ontario, London, Ontario N6A 587. Received 23*
348 rather specific bismuth binding to nuclear components such as the nucleolus and to ribosomes (Locke and Huie, 1975b). The work reported here shows, on the contrary, that bismuth binds very readily to basic proand probably to phosphorylated teins, molecules, but not to nucleic acids. This paper describes the procedures that we have used to show the consistent binding of bismuth by certain cellular components, and is illustrated by the range of tissues that we have surveyed to confirm the generality of the reactions. The paper also describes in vitro test systems that suggest a rationale for the bismuth staining. We have found that bismuth binds mainly in two ways, either through amino and amidine groups which are sensitive to glutaraldehyde, or through phosphate which is glutaraldehyde insensitive. Whatever the exact mechanism of bismuth binding the staining procedure is useful for distinguishing numerous cell components. Materials and Methods Organisms and cells tested. The bismuth staining was first used on Calpodes ethlius (Lepidoptera, Hesperiidae) larvae to show the Golgi complex beads (Locke and Huie, 1975a, 1976a) and the plasma membrane plaques involved in cuticle deposition (Locke, 1976). In the course of these studies it was noticed that the staining procedure could be adapted to show nucleoli (Locke and Huie, 1975b) and the contents of neurosecretory vesicles believed to contain biogenic amines, suggesting that bismuth staining might be generally useful for light and electron microscopy. We therefore tested the procedures that had been optimized for Calpodes upon a range of cell types from a variety of organisms from protozoa to mammals. Bismuth staining fbr light microscopy. A variety of fixatives can be used to allow demonstration of the nucleolus, including Carnoy, 70% ethanol, formaldehyde and osmium tetroxide. Glutaraldehyde greatly reduced the bismuth staining of components resolvable by light microscopy. The procedure below demonstrated nucleoli in whole tissues and in dewaxed sections. It was developed using Calpodes epidermal nuclei as test objects to find the optimal conditions of
time, temperature, pH, concentration, etc., for both fixation and staining. The use of black sulphides and haematoxylin lakes to visualize the location of heavy metals for light microscopy greatly reduces the labour needed to develop stains for electron microscopy. Schedule for bismuth staining of’ nucleoli
I to several hours, Fix in formaldehyde cold or at room freshly prepared from temperature paraformaldehyde with 0.05 M cacodylate buffer at pH 7.4 + hr-several Wash in 0.05 M Na hours cacodylate-HCI buffer with IO:/‘, sucrose, pH 7.4 Wash, 0.1 M triethanolamine-HCI pH 7.0
Bismuth staining solution
I hr at room temperature on a gentle rotary shaker
Preparation of staining solution: Solution A. Bismuth oxynitrate (Alpha Inorganics, Beverley, Mass.). Dissolve 400 mg sodium tartrate in 10 ml of I N NaOH. Add dropwise to 200 mg bismuth oxynitrate (BiONOs. HzO, also called bismuth subnitrate or basic nitrate. There is no advantage in preparing fresh bismuth nitrate from bismuth metal). Solution B Triethanolamine buffer. 0.2 M triethanolamine-HCI buffer. Adjust pH to 7.0. Bismuth staining solution. Add sol. B to sol. A in a 2:l to 5:l ratio. Readjust pH with HCI to 7.0. The more dilute solution is stable for I week at room temperature. The stronger solution is only stable for a few hours and then only at pH 8. Wash, 0.1 M triethanol30 min several amine-HCI pH 7.0 changes Either ammonium sulphide, several drops to final wash or stain for several minutes in haematoxylin Wash in distilled water, dehydrate thoroughly and mount. The brown/black colour of nucleoli is stable indefinitely after thorough dehydration
Bismuth staining for electron microscopy. The procedure was similar to that for light microscopy but used only fixation procedures known to give good ultrastructural preservation. The results varied dramatically with fixation; many, but not all, components that stained after formaldehyde did not do so after glutaraldehyde. Five per cent glutaraldehyde in 0.05 M cacodylate buffer at pH 7.4 with 4 % sucrose for 1 hr at room temperature was routinely used to block all but the characteristic glutaraldehyde insensitive bismuth staining. After staining in the bismuth solution, the tissue was post fixed in 1% osmium tetroxide at pH 7.2 in 0.05 M cacodylate with 4% sucrose before dehydration and embedding in resin. Osmium tetroxide can only be used as a primary fixative with difficulty since the buffers used during staining remove most of the osmium left in the tissue by fixation. There is no such problem using osmium tetroxide after staining. Some bismuth stained tissues were prepared without osmication to obtain sections in which the electron scattering was almost solely that introduced by the stain. Sections of such material as thick as 500 nm could usefully be observed at 100 kV and are now also being used for HV studies. Tissue or sections can becounterstained in uranyl acetate (Locke and Krishnan, 1971; Locke et al., 1971) without interfering with the bismuth stain, but the bismuth staining components are then easily overlooked. Enzyme digestions used to test the specificity of bismuth staining. The penetration of RNase
and DNase into tissue was first tested after various fixation procedures using pyronin staining and the Feulgen reaction as indicators of effectiveness. Glutaraldehyde fixed tissues tended to exclude RNase and left aldehydes in the tissue which interfered with the Feulgen reaction. Good enzyme penetration and satisfactory preservation for electron microscopy was obtained with formaldehyde fixed tissues washed for 2-3 hr in cacodylate buffer at pH 7.4 with 10% sucrose followed by 0.1 M Tris-HCl buffer pH 7.2 before incubation in either an enzyme solution or a buffer control solution at room temperature for 60-75 min. DNA digestion was carried out in 2 mg/ml. DNase (Sigma Chem. Co., St Louis, Missouri) in 0.1 M Tris-HCl buffer at pH 7.2. RNA digestion was carried
out with either RNase (ICN Pharmaceuticals, Cleveland, Ohio) or RNase TZ (Sigma Chem. Co., St Louis, Missouri) at a concentration of 2 mg protein to 1 ml of 0.1 M Tris-HCl buffer pH 7.2. The effectiveness of the DNase was determined by the Feulgen reaction. Samples were hydrolysed (8 N HCl, at room temperature for 30 min), washed with distilled water, stained in Schiff’s reagent and counterstained in 0.5 % alcoholic fast green. This DNase treatment made all nuclei Feulgen negative and electron microscopy showed them to be missing much of the component that can be stained by uranyl acetate upon the section. The effectiveness of RNase was determined by staining with freshly prepared methyl green-pyronin Y for 1 hr at room temperature. This RNase treatment removed all pyronin staining material and em observations showed the ribosomes to be swollen with diffuse outlines. In vitro testing of pure reagents and cell fractions for bismuth binding. Pure reagents and cell fractions were spotted onto cellulose strips (Gelman, sepraphore) for electrophoretic separation or immediate reaction. The strips were fixed in aldehyde vapour overnight at room temperature and then treated in the same way as tissue for light microscopy. The absolute amount of test reagent that survives fixation and is available for staining is unknown so that results can only be qualitative or roughly quantitative if fixation has to be used. The reaction of the spots with bismuth usually gave an unequivocal qualitative result which could be quantified by clearing the strip and measuring the density with a densitometer as is done for disc gel electrophoresis. (Acrylamide gels cannot be used as they react with bismuth.) It is surprising that this sensitive and simple procedure has not been more widely used to investigate the mode of action and specificity of staining molecules. The method has about the same sensitivity as Coomasie blue for detecting protamine and histones.
Results I. Cell components binding bismuth 1. The nucIeus Bismuth staining for light microscopy sharply delineated nucleoli in the whole mount of
Calpodes epidermal cells shown in Fig. I. Electron microscopy of these cells showed bismuth binding to the fine grained material forming the bulk of the nucleolus and also to several granular components (Fig. 2). This pattern of nuclear staining was confirmed in numerous animal cell types (Figs. 3, 7). The granular components were particularly clear in nuclei from mouse pancreas (Figs. 3,4, 5). The interchromatin granules are about 20 nm in diameter and are always present in euchromatic regions, often clustered away from the nuclear envelope (Fig. 4). The interchromatin granules appeared to have a lighter center in some orientations as though the bismuth had stained a cylinder of material. The perichromatin granules are about 50 nm in diameter and sometimes appeared to be reticulate (Fig. 5). Nucleoli are stained by very fine grains often showing the reticulate form in two densities, perhaps corresponding to the glutaraldehyde sensitive and insensitive regions (Fig. 6). All nuclei were
found to stain with bismuth in this manner. In addition, bismuth stained groups of irregular filaments and parts of the synaptinemal complex in meiotic cells from mouse testis. Fig. 8 shows a uranyl-stained mouse pancreas nucleus for comparison with similar material stained by bismuth (Fig. 3). In some respects bismuth staining is complementary to uranyl. Both stain the nucleolus and inter- and perichromatin granules but bismuth does so much more intensely. They differ in that uranyl stains the chromatin which is completely unaffected by bismuth. The characteristic feature of bismuthstained nuclei is that few components are stained but these few stain with great precision. 2. The cytoplasm-ribosomes Bismuth stained free and bound ribosomes and also small particles in mitochondria that are presumed to be ribosomes. There was usually little or no staining of membranes,
Key fo figure captions
A AC Acr b BL C CF Ch DL H HD IC ICG mit MT N Nu PCG KER T
Annulus Alveolar compartment at a ciliate surface Acrosome GC bead Basal lamina Cuticle Cleavage furrow Chromatin Post-acrosomal dense lamina Haemocoel Hemidesmosome Interchromatic region Interchromatin granules Mitochondria Microtubules Nucleus Nucleolus Perichromatin granules Ri bosome Rough endoplasmic reticulum Tonofibril
Fig. 1. Bismuth staining of nucleoli for light microscopy. Whole mount of Calpodes epidermis and cuticle showing nucleoli. Treatment: formaldehyde, bismuth, ammonium sulphide. x 1150. Fig. 2. Bismuth staining of nuclei for electron microscopy. Calpodes epidermis as in Fig. 1 but prepared for electron microscopy. Bismuth stains the nucleolus, nuclear granules and ribosomes but not chromatin. Treatment: formaldehyde, bismuth, 0~04. x 20,ooo.
mitochondrial matrix, the contents of the cisternae of the ER or Golgi complex, the densely packed protein of secretory vesicles or microfibers and microtubules. Bismuth staining of ribosomes resulted in irregular grainy aggregates rather than the regular outlines seen after uranyl acetate. This was not because the treatment had disrupted the structure of the ribosomes since the usual shape could be made out after subsequent uranyl staining. The bismuth may therefore be binding to only some components of the ribosomes or the binding may perhaps cause a coarse grained deposition that has no counterpart in ribosomal structure. 3. Membrane specializations Plasma membrane plaques. At the surface of
the plasma membrane of an insect epidermal cell engaged in secreting either fibrous cuticle or the outer epicuticle, there are localized densities on the cytoplasmic surface, somewhat resembling hemidesmosomes (Locke, 1976, 1967). These stain by uranyl acetate and lead citrate upon the section (Fig. 9) but this procedure does not distinguish the plaques from many other membrane associated components. One of the original objectives in testing the staining properties of several heavy metals was to find a method for staining insect plasma membrane plaques in a characteristic way in order to follow their formation, involution and function in cuticle
deposition. Bismuth staining of tissue gave good contrast to the plaques without staining other components in the apical secreting region. The typical appearance of bismuthstained plasma membrane plaques from insect epidermis is shown in Figs. IO and 1I. They are usually (Fig. 1I ), but not always (Fig. 13) at the tips of microvilli. They also occur at secretory surfaces in other organisms such as the lining of crayfish digestive glands (Fig. 16) and epithelia of annelids and mollusts (Fig. 15) but not at the tips of all microvilli; for example they did not stain at the microvillar tips lining the vus deferens and small intestine of mice. The form of the plaque varies with the plasma membrane. It may be on a barely raised surface (C&odes epidermis secreting tracheal cuticle, Fig. 13), or it may follow the rounded (Orconectes digestive gland, Fig. 16) or flat topped (Spirorbis epithelia Fig. 15) contour of microvilli. trast
is due entirely to the bismuth staining. Without staining only the plasma membrane shows up due to its osmiophilia (Fig. 12). Hemidesmosomes (Fig. 14) on the basal face of these same epidermal cells did not react with bismuth in spite of their superficial structural similarity to the plaques on the apical face. We concluded from these observations that there are regions of speciahzation on the cytoplasmic face of plasma
Figs. 3-7. The nuclear components
stained with bismuth.
Fig. 4. Bismuth staining of the interchromatin granules. Treatment: formaldehyde, bismuth, 0~04. x 85,000.
Fig. 5. Bismuth staining of perichromatin formaldehyde, bismuth, 0~04. x 160,000.
Fig. 6. Bismuth staining of the nucleolus. hyde, bismuth, 0~04. x 39,000.
Mouse Sertoli cell. Treatment:
Mouse Sertoli cell. Treatment: formalde-
Fig. 7. Bismuth staining of the nucleolus. Crayfish x-organ neurosecretory Treatment: short glutaraldehyde fixation, bismuth, 0~04. x 33,OQO.
Fig. 8. The nucleus stained by uranyl acetate for comparison pancreas. Treatment: formaldehyde, 0~04, section stained Compare with Fig. 3. x 24,000.
membranes, that can be distinguished by bismuth staining in away that is not possible with conventional surface staining of sections. In order to get a better idea of the generality and usefulness of this aspect of bismuth staining, numerous cell types having furry cytoplasmic coatings to the plasma membrane were observed after bismuth staining.
with bismuth. Mouse with uranyl acetate.
Junctions und reluted structures. At least some junctions between cells bind bismuth. The staining was confined to the junctions close to the apical face of epithelia in the position of tight and intermediate junctions. The pattern was similar in Coelenterates (Fig. IS), annelids (Fig. 17), vertebrates (Fig. 19) and insects although these junctions are not all
exactly alike morphologically. The stain was specific for the junctional region and was absent from nearby surface plasma membranes, ER membranes and other types of junction such as septate desmosomes. The stain was in the form of a fine-grained precipitate on the cytoplasmic face of the junction and may have some periodicity. Some synaptic junctions also stained with bismuth. Fig. 20 shows the characteristic deposit at pre- and post-synaptic junctions in nervous tissue from a crayfish X-organ. At some double and multiple junctions the bismuth is localized in patches on the cyto-
Figs. 9-16. Bismuth staining
plasmic face with a small separation from the membrane. A very similar staining pattern was observed in Calpodesneuromuscular junctions, but here the bismuth was all on the muscle cell membrane (Fig. 21). In insect muscle insertions the tonofibrils from the cuticle pass into crypts in the epidermis where they attach to bundles of microtubules traversing the cell to the basal face where they associate with junctions to the muscle. The cytoplasmic face of these tonofibrillar crypts binds bismuth with an appearance very similar to intermediate junctions (Fig. 22).
Fig. 9. The plaques at the tips of microvilli on the apical face of an epidermal cell secreting fibrous cuticle. Calpodes integument. Treatment: glutaraldehyde, 0~04, section stained with uranyl acetate and lead citrate. There are densities at the tips of these wedge-shaped microvilli but no specific staining. x 23,000. Figs. 10, 11. The plaques stained by bismuth. Preparation identical to Fig. 9 but stained only with bismuth. Density is confined to the cytoplasmic face of the tips of the microvilli. Treatment: glutaraldehyde, bismuth, 0~04. Fig. 10. x 49,000; Fig. 11, x 130,000. Fig. 12. The density due to osmium alone. Preparation identical but without bismuth. The plaques are not stained. x 130,000. Fig. 13. Plaques at the surface of the tracheal Treatment: as Figs. 9 and 10. x 130,000. Fig. 14. Hemidesmosomes do not stain Treatment: as Figs. 9 and 10. x 81,000.
to Figs. IO and
which has no microvilli. Cnlpodes
Fig. 15. Plasma membrane plaques from annelid epidermis. Spirorbis Bismuth also stains the mucus holding these microvilli together. Treatment: and 11. x 97,000. Fig. 16. Plasma membrane plaques upon Treatment: as Figs. 10 and 11. x 87,000. Figs. 17-21. Bismuth
oral filter. as Figs. 10
Fig. 17. There is a fine grained bismuth deposit on the cytoplasmic face of the adhering zonula. Spirorbis oral wreath epidermis. Treatment: glutaraldehyde, bismuth, 0~04. x 82,000. Fig. 18. Adhering x 81,000.
Fig. 19. Adhering ronula at the level of the tight junction small intestine. Treatment: as Fig. 17. x 150,000. Fig. 20. Synapse from crayfish x-organ. The bismuth the cytoplasmic face. Treatment: as Fig. 17. x 80,000.
Treatment in epithelium
is slightly away from
Fig. 21. Neuromuscular junction from Calpodes larva. There is a bismuth deposit on the cytoplasmic face of the muscle plasma membrane but only very slight deposits on the presynaptic face. Treatment: as Fig. 17. x 46,000.
Contractile and moving membranes. An early indicator of cell division is the appearance of a thickened plasma membrane in the cleavage furrow. It becomes the membrane of the mid-body before finally constricting the cell into two. This membrane stained with bismuth (Fig. 23). It seemed possible that bismuth staining might be associated with moving or contracting membranes in a general way. A special study was therefore made of functionally comparable membranes -the acrosome at the time that it spreads around the nucleus and the annuli of the plasma membrane-as they move in relation to spermatogenesis in mouse testis. The annulus is a dense mass attached to the plasma membrane in a ring round the axial complex. It moves backwards from behind the nucleus with the separation of the mid-piece from the tail (Fawcett et al., 1970; Phillips, 1974). Fig. 24 shows an early stage in the formation of the annulus and its reaction with bismuth. There is a fuzzy bismuth staining mass corresponding to the annulus anlagen. Nearby structures of equal or greater unstained density do not show this reaction although they may have some osmiophilia and may stain intensely with uranyl salts. The acrosome is a sac that flattens around the anterior face of the sperm nucleus as it accumulates material from the Golgi complex (Phillips, 1974; Baccetti and Afzelius, 1976). The margins of the sac appear to adhere to the nuclear envelope through several structures, one of which survives formaldehyde fixation and reacts with bismuth. Figs. 25 and 26 show profiles of the rim of the acrosome with a granular patch of bismuthstained material. The density is on the cytoplasmic face and has the same sort of texture and appearance as the bismuth stained cell junctions. We conclude that bismuth staining can be used to distinguish some sorts of membrane associated cytoplasmic structures from others in a way that is not usually possible by the conventional staining of sections. Specializedsurfaceplasma membranes. In two cell types (Paramecium and mammalian sperm) bismuth stained large areas of surface membrane in a very striking way. In Paramecium the plasma membrane folds inwards to form an alveolar compartment almost com-
pletely separated from the surface. The inner half of this alveolar compartment has a membrane with a surface coat that stains with bismuth (Fig. 27). The bismuth binding material is thus arranged in longitudinal strips between cilia corresponding in position to the calcium sensitive conducting membranes that have been postulated to coordinate ciliary beat (Eckert, 1972). The post-acrosomal dense lamina is a specialized region of plasma membrane around the hind part of the sperm head (Fawcett, 1970). The cytoplasmic face of the membrane in this region binds bismuth intensely in forming and mature mouse sperm (Fig. 28). Unique structures only observed after bismuth staining. The Golgi complex beads. The region
between the rough endoplasmic reticulum and the Golgi complex was studied after bismuth staining in a variety of cell types in an attempt to find a marker for the exit gate or g,ttes from the ER (Locke and Huie, 1975a, 1976a, c). The smooth surface of the rough endoplasmic reticulum making the forming face of the Golgi complex has beadlike structures arranged in rings at the base of transition vesicles. The beads can be seen after bismuth staining in all arthropod cell types (Fig. 29). They are IO-12 nm in diameter and are separated from the membrane and one another by a clear halo giving them a center to center spacing of about 26 nm. They also occur in vertebrates but are difficult to resolve there because they do not stain with bismuth (Locke and Huie, 1976~). The beads seem to be a general feature of Golgi complexes but the specific staining by bismuth which led to their discovery seems so far to be confined to arthropods. The reaction associated with the outer tubules of sperm tails. The rationale for looking for
bismuth staining structures in the GC-RER transition region depended upon the finding that there is an energy dependent step in the movement of secretion from the RER to the GC (Jamieson and Palade, 1968). An energydependent process might be expected to involve polyphosphates that could react with bismuth if they were to be held in position by fixation. If this rationale for the staining of the GC beads is correct, then it ought to be possible to localize other energy-dependent processes.
With this in mind we examined the motor apparatus (Fawcett, 1970) in bismuth-stained sperm tails, particularly where the axial complex is close to mitochondria. Figs. 30 and 3 1 show that there is a bismuth-stained region next to the outer doublet microtubules. It is present only in the mid-piece and anterior part of the principal piece and is absent from the end piece. The localization is exceedingly precise and reflects the sperms’ asymmetry by corresponding in amount to the size of the outer dense fibers. Thus, under the conditions described, bismuth stains a range of cell structures, some specifically, in a way that may make the stain useful for developmental studies. In an
attempt to increase the specificity of some of the reactions we investigated the effects of fixation in more detail. 11. Glutavaldehyde staining
as (I srlertiw
Glutaraldehyde blocks some staining that is present after formaldehyde fixation. Table I lists the sensitivity of various components to staining after glutaraldehyde. Bismuthstained components can be grouped into two categories, those that stain after formaldehyde but not after glutaraldehyde and those stained after formaldehyde that continue to stain after glutaraldehyde. We conclude from this that two kinds of chemical reaction may
Fig. 22. The apical adhesions to cuticular tonotilaments. Calpodes muscle insertion. Section plane normal to tonofilaments passing from the cuticle into apical infolds. There is a bismuth deposit on the cytoplasmic face. These cells are filled with microtubules which traverse the cell to the muscle adhesions on the basal surface. Treatment: glutaraldehyde, bismuth, 0~04. x 80,000. Fig. 23. Bismuth staining of cleavage furrows during cytokinesis. Mouse spermatocyte. Treatment: formaldehyde, bismuth, 0~04. Ribosomes are also stained in this formaldehyde fixed material. x 76,000. Fig. 24. Bismuth staining of the annulus during spermatogenesis. Mouse spermatocyte. Treatment: as for Fig. 23. The fuzzy material on the cytoplasmic face of the annulus stains with bismuth. x 87,000. Figs. 25,26. Bismuth staining of material (J) on the cytoplasmic face of the leading edge of the acrosome during mouse spermatogenesis. Treatment: as for Fig. 23. Fig. 25. ~78,000; Fig. 26, x 160,000. Fig. 27. Ciliate plasma membranes. The inner face of the alveolar compartment in Paramecium binds bismuth on its outer surface. The reaction stops abruptly where the alveolar membrane lies adjacent to the outer plasma membrane. Treatment: glutaraldehyde, bismuth, 0~04. x 39,000. Fig. 28. Bismuth is a specific stain for the post-acrosomal sperm. Treatment: formaldehyde, bismuth, 0~04. x 79.000.
Fig. 29. Bismuth is the only staining method known to show up the Golgi complex heads. Crayfish x-organ neurosecretory cells. Treatment: glutaraldehyde bismuth, 0~04. x 124,000. Figs. 30, 31. Bismuth staining of a sperm tail component on the outer face of the peripheral doublets. Fig. 30 TS and Fig. 31 LS mouse sperm tail. Treatment: formaldehyde, bismuth, 0~04. x 134,000. Fig. 32. Glutaraldehyde fixation blocks the bismuth staining of many components such as nucleoli and ribosomes, but not others such as inter- and perichromatin granules. Mouse Sertoli cell. Treatment: glutaraldehyde bismuth, osmium. x 31,tJOO. Fig. 33. Bismuth staining for differentiating various kinds of neurosecretory vesicles. Those presumed to contain biogenic amines stain, others do not. Neither kind of neurosecretory vesicle stains after glutaraldehyde fixation. Calpodes. Corpus cardiacum. Treatment: formaldehyde, bismuth, 0~04. x 29,000.
Table 1. The effeect of glutaraldehyde upon structures that stain with bismuth after jbrmaldehyde
After formaldehyde fixation all the structures below are stained by bismuth Nucleolus Ribosomes Neurosecretory granules (believed to contain biogenic amines) Paramecium inner alveolar membrane Mouse sperm dense lamina Cleavage furrow Acrosome leading edge Mouse sperm annulus Plasma membrane plaques Tonofibril membrane Apical junctions Some synapses and neuromuscular junctions Sperm tail doublets Golgi complex beads Interchromatin granules Perichromatin granules Chromatin after DNase
Bismuth staining after glutaraldehyde fixation Slight to _
-. _ _ + + + ++ ++ ++ ++ ++ _
be involved. For practical purposes glutaraldehyde leaves tissues in a state that gives very clean and unequivocal staining. After formaldehyde the reaction has the appearance of being less specific because of the larger number of components stained. Alcoholic and acidic fixatives for light microscopy, such as Carnoy, leave tissue staining as it does after formaldehyde, that is, they can be used to demonstrate nucleoli. Prolonged formaldehyde fixation causes proteins to be permanently cross-linked through methylene bridges, but the initial reaction is reversible (Hayat, 1975, p. 86). After mild formaldehyde fixation (hours at RT) aldehydes were removed by washing for hours to days. As this happened, the background of bismuth staining increased whereas prolonged fixation (days at RT) resulted in the bismuth staining being greatly and irreversibly reduced as happened after glutaraldehyde. It seemed as though the initial reversible formaldehyde reaction and that of glutaraldehyde were both concerned with the groups that bind bismuth. Glutaraldehyde fixes by the irreversible cross-linking of the amino groups of proteins (Hayat, 1975, p. 86),
suggesting that kind of bismuth basic groups of been confirmed other molecules IV).
the glutaraldehyde sensitive staining may be through the proteins. This hypothesis has in spot tests on proteins and with amino groups (section
111. The nature qf the tissue reactions. podes epidermal nuclei as test objects
The nucleoli in Calpodes epidermis are more clearly delineated by bismuth followed by either haematoxylin or ammonium sulphide than by any procedure that we know (Fig. I ). Such preparations also show a highly selective pattern of staining for electron microscopy making them suitable test objects to try to determine the nature of the groups reacting with bismuth. It seemed possible that in some nuclear components the bismuth might be binding to the exposed phosphate of a nucleic acid, since it had been claimed that chromatin and RNA stained with bismuth in onion root tip nuclei, and bismuth precipitated DNA and RNA in vitro (Albersheim and Killias, 1963). Our staining of ribosomes and nucleoli agreed with this notion. We therefore tested the effect of nucleases upon the ability of the nucleolus to stain with bismuth. I. The t$Fect of RNase upon the staining of nuclear components by bismuth Formaldehyde fixed tissue was treated with beef RNase and TZ RNase (which both gave the same result) and then either observed in whole mounts after pyronin Y staining to determine the effectiveness of the RNase in removing nucleolar and ribosomal RNA, or prepared for electron microscopy. Forty minutes exposure to RNase completely removed all the material stained by pyronin. A similar incubation in buffer solution caused no loss of staining. Electron microscopy of uranyl stained sections of this RNase treated tissue showed diffusely stained ribosomes which were grossly puffed up (Fig. 34). There is therefore little doubt that the RNase has had an effect. In spite of this, the bismuth continued to stain the puffed up remnants of ribosomes (Fig. 35), the nucleolus (Fig. 36), the perichromatin granules and interchromatin granules (Fig. 37). It is also possible to see that the morphology of the CC beads, unlike the ribosomes, is unaffected by RNase and that they also continue to be stained by
Fig. 34. RNase affects ribosomal structure after formaldehyde fixation. Calpodes epidermis. Treatment: F, RNase, Glut, Bi, OS. Sections stained in uranyl acetate to show the puffed up appearance of the ribosomes (I). The uranyl staining overshadows thar due to bismuih but the beads can still be made out and are presumed not to contain RNA. Compare with Fig. 35. x 160,000. Fig. 35. RNase disrupts ribosomes but not beads in formaldehyde fixed tissue. Calepidermis. RNase causes a gross alteration of ribosomal structure without reducing bismuth staining or affecting bead structure. Both ribosomes (r) and beads (b) still bind bismuth, suggesting that the bismuth is not binding to RNA, and that the beads do not contain RNA. The bismuth binding material in ribosomes is Glut. sensitive and is not the same as the material binding bismuth in the beads which is Glut. insensitive. Treatment: F, RNase, Bi, OS. x 150,000.
bismuth (Figs. 34, 35). This continued staining of RNA containing structures after RNase digestion suggested that the bismuth was not staining RNA. 2. The effect of DNase upon the staining of nuclear components by bismuth Tissue was treated with DNase and then either observed in a whole mount by light microscopy after Feulgen staining to determine the effectiveness of the DNase in removing nuclear DNA, or processed for electron microscopy. Glutaraldehyde could not be used to fix tissue for these experiments because it leaves residual aldehyde that reacts with the Schiff’s reagent making it impossible to determine the effectiveness of the DNase.
Formaldehyde fixed material gave excellent Feulgen preparations which were consistently and completely negative in cell nuclei after treatment with DNase at room temperature for 75 min. Electron microscopy showed much material missing from these nuclei. There is therefore little doubt that the DNase has heen effective. Although DNA is inferred to be absent from these nuclei, the nucleoli, perichromatin granules and euchromatin granules continue to stain with bismuth (Figs. 38, 39). Removal of the DNA also caused other nuclear components to stain such as the outer cortex of the nucleolus and the heterochromatin. This extra staining induced by DNA removal was blocked by postfixation in glutaraldehyde.
We concluded from these experiments that in fixed cells neither category of bismuth staining involves nucleic acids. IV. The staining of isolated cell components and molecules The lack of bismuth staining by tissue nucleic acids has been confirmed by spot tests. 2 ~1 of 1% RNA (To&a yeast, Sigma) and DNA (calf thymus, Sigma) in water and in 2% gelatin were spotted on cellulose strips and fixed at room temperature overnight in formaldehyde vapour or tested without fixation. The object of fixing and particularly of fixing in gelatin is two-fold: to treat the test molecules to the same conditions as they would have had in fixed tissues and to prevent the loss of the test molecules from the strip by cross-linking them. However, fixation can be self defeating when fixative and bismuth are both reacting through the same groups. For this reason both fixed and unfixed test spots were reacted in the bismuth solutions used to stain tissues. Fixed gelatin alone gave only a very faint brown reaction. Although it was routinely used to increase the probability of
cross-linking the test molecules, it was unnecessary for nucleic acids. DNA and RNA were readily visualized by methylene blue and pyronin staining but developed only the slightest trace of brown/black color (Fig. 40). We concluded that nucleic acids are not demonstrated under the conditions used to prepare bismuth stained tissue for light and electron microscopy. This result focused attention upon certain kinds of protein as probable causes of bismuth staining. In particular, if the amino groups of basic proteins are involved it could account for the block to the nucleolar (and other) reactions by glutaraldehyde and the staining of DNase treated chromatin, which could have been brought about by the exposure of basic groups formerly neutralized by DNA. With this in mind a number of basic proteins and similar molecules were tested for their ability to bind bismuth. In contrast to gelatin and bovine serum albumen, histones and protamines reacted very strongly with bismuth (Fig. 40). The weak reaction of fixed neutral proteins ruled out peptide links and suggested that the
Figs. 36, 37. RNase does not affect the bismuth staining of ribosomes or nuclear components. Calpodes epidermis. Treatment: formaldehyde, RNase, bismuth, 0~04. Although ribosomal structure is grossly distorted (Figs. 34, 35), the staining of the nucleolus, interchromatin and perichromatin granules is not reduced. Compare with Fig. 2 for control, not treated with nuclease. Fig. 36, x 47,000; Fig. 37, x 74,000. Figs. 38, 39. DNase causes chromatin to stain with bismuth without reducing the staining of any other nuclear components. Cafpode$ muscle cell. Treatment: formaldehyde, DNase, bismuth, 0~04. Tissue containing no DNA continues to stain with bismuth. The extra staining of chromatin lacking DNA is presumably due to bismuth binding to protein at the sites formerly occupied by DNA. Compare with Fig. 2 control, not treated with nuclease. Fig. 38, x 30,000; Fig. 39, x 81,000. Fig. 40. Spot tests show that basic proteins bind bismuth but not nucleic acids or neutral proteins. The bismuth-staining procedure does not remove the nucleic acids which continue to stain intensely with methylene blue. The gelatin was in 2 % solution, all others were 1 %. Treatment: formaldehyde vapour 48 hr; formaldehyde fixative solution 2 hr; bismuth staining as for tissue. Fig. 41. Biogenic amines stain with bismuth. Since serotonin and tryptamine react but indole does not, the binding is presumed to be through amino groups. The indole survives the procedure and can be visualized by its UV fluorescence. All reagents were I % aqueous solutions. Treatment: as Fig. 40. Fig. 42. Some phosphates bind bismuth even after glutaraldehyde fixation. All test spots were in 1 ‘A gelatin, ADP and AMP were 5 % solutions, the others 1%. Since adenosine does not stain, the other reactions are presumed to be due to phosphate. Glutaraldehyde does not reduce the reaction very markedly. Treatment: as Fig. 40.
tryptomine histone protomine
@ * indole
Table 2. Glutaraldehyde jixation blocks more of the bismuth reaction attributed to binding by amino groups than formaldehyde. The bismuth staining of histone and polyarginine is affected less than polyIysine by gtutaraldehyde as would be expected if bismuth binding is also through guanidine groups. AN reagents were I 0/Oin l”/, gelatin except polyIvsine at 0.5Y in water. Glutaraldehvde forms brightly colored complexes with amines *which interfere with a photographic presentation of the results (spermine and spermidine pink; serotonin, yellow; dopamine, orange). The density of brown stain due to bismuth has been estimated on a fivepoint scale
Density of bismuth staining after ____ Spermine Dopamine Serotonin Polylysine Polyarginine Histone
Formaldehyde +++++ ++++ +++ ++++ +++++ ++++
Glutaraldehyde Variable to ++ + + ++++ +++
guanidine (= amidine) groups of arginine or the amine groups of arginine and other amino acids might be reacting with bismuth. (Cobalt ammine coordination complexes are known and bismuth might form similar and equally stable bonds.) The very strong reaction of polylysine as well as polyarginine made amines a most likely candidate. If basic proteins react with bismuth through amine side chains then we should expect a similar reaction from polyamines such as spermine and spermidine. These molecules did indeed react intensely with bismuth. So did biogenic amines such as dopamine, tryptamine and Serotonin (5HT) (Table 2). Indole, having the same structure as tryptamine and serotonin but without the free amino groups, had no affinity for bismuth, showing that thereaction is through terminal amines and not through basic parts of the indole ring. The successful staining of these fixed molecules in vitro prompted a return to electron microscopy and a search for neurosecretory cells that might be expected to have vesicles containing biogenic amines. Fig. 33 shows neurosecretory cells of two kinds from the corpus curdiacum of Calpodes. One cell type has neurosecretory granules reacting with bismuth and the other type had no reaction. We take this
as confirmation that bismuth reacts with amines in tissues as well as in vitro and does so in a way that may make it useful for their localization. The previous finding (section II), that some bismuth staining is sensitive to fixing in glutaraldehyde, agrees with the conclusion above that this bismuth staining is through amino groups, particularly those on basic proteins. Molecules with free amino groups tested in vitro are also sensitive to glutaraldehyde fixation. Spot tests on serotonin, dopamine, spermidine and polylysine all show a great reduction in bismuth staining after glutaraldehyde fixation compared to formaldehyde (Table 2). There is less reduction in histones and polyarginine as would be expected if guanidine groups are only fixed at pHs higher than 9 as Hayat suggests (Hayat, 1975, p. 76). Prolonged fixation of tissues by glutaraldehyde might be expected to block guanidine groups but it had little effect upon the staining of components listed as glutaraldehyde insensitive (Table 1). It is therefore more probable that this staining is due to phosphate (see below) rather than guanidine groups. We concluded from these experiments that the glutaraldehyde sensitive bismuth staining is probably due to complexing with amine and guanidine groups, particularly those of basic proteins. The most likely cause of the glutaraldehyde insensitive bismuth staining seemed to be phosphate, since ferritin stains strongly with bismuth (Ainsworth and Karnovsky, 1972; Ainsworth er al., 1972) and bismuth in 0.1 N HsSG4 is precipitated by excess phosphate in vitro (Albersheim and Killias, 1963). Even after glutaraldehyde fixation we found that bismuth gave ferritin extra contrast and interfered with a search for GC beads in frog and mouse liver. In our original study describing the discovery of the beads we used bismuth in the belief that it would localize bound high energy phosphate marking the energy dependent step in transport through the GC. To determine if phosphate could be the cause of the glutaraldehyde insensitive bismuth reaction we tried to stain ATP, ADP, AMP, and phosphoenol pyruvate as well as other phosphates. Fig. 42 shows some of the results. All these molecules were fixed in the cellulose strips by formaldehyde, particularly
when mixed with gelatin, and they all reacted with bismuth. In contrast, similar molecules lacking the phosphate, such as adenosine or polyuridylic acid showed no such reaction. Nor could the staining be attributed to -OH (there is little difference between tryptamine and 5HT), -COOH (since polyglutamic acid was negative) or -SH (polycysteine was negative). There is therefore no doubt that organic phosphates of biological interest but not many other common groups, can react with bismuth under the conditions used to stain tissues. When these phosphates were fixed by glutaraldehyde the bismuth reaction was reduced little or not at all compared to molecules such as 5HT or polylysine (Fig. 42). Thus biological phosphates are a likely cause of the glutaraldehyde-insensitive bismuth staining. Discussion
We have developed a staining procedure that is equally suitable for light microscopy, for electron microscopy and for molecules and cell fractions isolated on certain types of gel. Electron microscopists have been slow to follow up the work of classical histologists in their use of heavy metal mordants, perhaps because of the effort involved in testing for tissue staining compared to the quick and easy but relatively non-specific section staining. As we have shown, light microscopy greatly reduces the labor needed to find the conditions for optimal binding of metals to cell components as a preliminary to their use as electron stains. Surprisingly, bismuth was one of the few metals that had not been tried as a mordant for haematoxylin. Gray (1954) does not list bismuth among the more than 15 useful metal mordants. Electron microscopists have also been slow to invert the
usual functions of electrophoresis and chromatography and to use these procedures to test the specificity of stains upon known isolated cell components and molecules under conditions similar to those used to stain tissues. We have shown how simple and informative this procedure can be for bismuth and work is now in progress upon staining by other heavy metals in common use. The generalization made here is that bismuth stains through amine and guanidine groups and that these can be blocked by glutaraldehyde. Staining not blocked in this manner may be through biologically important phosphates. Comments upon the significance of particular localizations can only be speculation without much more work, but the distribution of staining does agree with the generalization above. Chromatin stains in a glutaraldehyde sensitive way only after the removal of DNA and the exposure of basic proteins. Lysine and arginine-rich proteins occur in the nucleolus and ribosomes where bismuth staining is also glutaraldehyde sensitive. If interchromatin and perichromatin granules are sites for phosphorylation it would account for their glutaraldehyde insensitive staining. Greengard (1976) postulates a role for cyclic nucleotides and phosphorylated membrane proteins in the post synaptic actions of neurotransmitters. Phosphorylated proteins might be the cause of the bismuth reaction in synapses and also in the region of apical junctions if there is a parallel between synaptic andepithelial cell communication. The glutaraldehyde-insensitive staining of the CC beads and the sperm tail doublets could both relate to phosphates involved in energy transformation. Whatever the exact mechanism of staining, the procedure should have value for visualizing some cell components, particularly in thick sections for three-dimensional interpretations.
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