THE ROLE OF THE ENDOSPERM IN THE GERMINATION OF LEGUMES: GALACTOMANNAN,NITROGEN, AND PHOSPHORUS CHANGES IN THE GERMINATION OF GUAR (CYAMOPSIS TETRAGONOLOBA; LEGUMINOSAE)

The endosperm of Cyamopsis tetragonoloba (“guar”) contains 41 % of the dry weight and 45 % of the acetone-insoluble-solids of the seed, but only 3–11 % of the nitrogen and phosphorus. At least 75 % of the acetone-insoluble-solids of the endosperm is galactomannan, only about 12% being accounted for as pentosan, pectin, protein, phytin, ash, and dilute-acid-insoluble residue. During a five-day germination period at 30 C, all of the galactomannan and all but 5 % of the dry weight of the endosperm disappeared, being translocated to the cotyledons. About ⅓ of the nitrogen and phosphorus of the endosperm plus seed coat were also translocated. After a 36-hr lag, the accumulation of the nitrogen and acetone-insoluble-solids in the seedling axis were linear, while the total dry weight and phosphorus showed a rapid increase followed by a slower accumulation during the five-day period. In the cotyledons, the dry weight temporarily increased, but the acetone-insoluble-solids, nitrogen and phosphorus showed only a net decrease after 84, 36 and 36 hr, respectively. Scanning election micrographs of dry-fractured and sectioned endosperm show that the bulk of the endosperm is a solid mass of galactomannan with essentially no cell lumina; a several-cell layer (“aleurone”) of thick-walled cells of similar structure is metabolically active.     

Subcellular localization of type I thionins in the endosperms of wheat and barley

Summary Thionins are cysteine-rich polypeptides of about 5,000 Da. Localization at the subcellular level of type I endosperm thionins has been carried out by immunogold labeling, using an antibody that recognizes type I thionin variants. In developing wheat and barley caryopses, sectioned at different times between 13 and 24 days after flowering, this type of thionins was only detected around protein bodies from cells of the starchy endosperm, using light microscopy. Electron microscopy revealed that these proteins were located in electron-dense spheroids in the periphery of protein bodies, at the earlier stages, whereas later the label appeared also as a thin layer around these organelles.Abbreviations DAF days after flowering - RER rough endoplasmic reticulum     

Adaptation of the Millon, Sudan Black B, and Periodic Acid-Schiff Technics for Block Staining of Insect Tissues

Spermatophores and reproductive systems of the beetle, Lytta nuttalli Say, fixed in Bouin's aqueous picroformol or buffered 10% neutral formol were stained in toto by the Millon, Sudan black B and periodic acid-Schiff reactions as follows. Millon: after excess fixative is removed in 70% ethanol, specimens are brought to water, stained in Millon's reagent at 60 C for 1 hr, rinsed in 2% aqueous nitric acid at 40-50 C, dehydrated rapidly, cleared, embedded and sectioned as usual. Sudan black B: specimens are taken to absolute ethanol, stained in a saturated solution of Sudan black B in absolute ethanol at room temperature for 24-48 hr, rinsed and cleared in xylene, embedded and sectioned. PAS: specimens are brought to water, oxidized in 0.5 aqueous HIO₄ at 37 C for 30 min, washed in 2 changes of water, stained in Schiif reagent at room temperature for 1 hr, rinsed in 3 changes of 0.5% aqueous potassium metabisulfite, washed in running water for 10-15 min, dehydrated, cleared, embedded and sectioned. All 3 methods produced their characteristic staining in specimens up to 3 mm thick     

Transgenic sorghum with improved digestibility of storage proteins obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Development of transgenic plants with modified seed storage protein composition and increased nutritive value is one of the most promising areas of genetic engineering. This task is especially important for sorghum—a unique drought tolerant cereal crop that is characterized, however, by a relatively poor nutritive value in comparison with other cereals. It is considered that one of the reasons of the low nutritive value of the sorghum grain is the resistance of one of its seed storage proteins, γ-kafirin, located in the outer layer of endosperm protein bodies, to protease digestion. Using Agrobacterium-mediated genetic transformation, we obtained transgenic sorghum plants (Sorghum bicolor (L.) Moench) harboring a genetic construct for RNAi silencing of the γ-kafirin gene. In the T₁ generation, the plants with almost floury or modified endosperm texture of kernels were found. In these kernels, the vitreous endosperm layer has been reduced and/or covered by a thin layer of floury endosperm. In vitro protein digestibility (IVPD) analysis showed that the amount of undigested protein in transgenic plants from the T₃ generation was reduced by 2.9–3.2 times, in comparison with the original non-transgenic line, and the digestibility index reached 85–88% (in comparison with 59% in the original line). In T₂ families, the plants combining high IVPD with vitreous endosperm type were found. In the electrophoretic spectra of endosperm proteins of transgenic plants with increased digestibility, the proportion of 20 kD protein that is encoded by the γ-kafirin gene, was significantly reduced, in comparison with the original non-transgenic line. HPLC analysis showed total amino acid content in two out of the three studied transgenic plants from the T₂ generation was reduced in comparison with the original non-transgenic line, while the lysine proportion increased by 1.6–1.7 times. The mechanisms conditioning improved digestibility of storage proteins in transgenic plants are discussed. The results of experiments demonstrate that it is feasible to develop sorghum lines combining high protein digestibility and vitreous endosperm that has a high breeding value.     

Studies on the Proteinaceous Subcellular Particles in Rice Endosperm: Electron-Microscopy and Isolation

Electron-microscopic examination of rice endosperm revealed the existence of protein-aceous subcellular particles, 1 to 4 µ in diameter and spherical or oval in shape. Isolation of the particles was effected by differential centrifugation in density gradient medium after mechanical or enzymic disintegration of endosperm cells. The isolated particles were predominantly composed of protein, and residual constituents were mainly lipid and carbohydrate. Their shape and behaviors were similar to those found in the endosperm. These facts show that the subcellular particles concerned are “protein bodies” There seemed to be several kinds of protein bodies different with respect to their protein and lipid contents.     

A Celloidin Infiltration-Frozen Section Sequence for Enhanced Preservation of Phosphatases in Bone

The effect of the following embedding procedures on the acid and alkaline phosphatase content of decalcified mouse tibiae has been studied: embedding in 23% gelatine for 18 hr at 37° C, embedding in paraffin wax in vacuo for 1 hr at 58° C, and impregnation with 4% celloidin in diethyl ether and ethanol at 4° C for 2-3 days. Unsupported tissues were also used to demonstrate these enzymes for comparison with the above procedures. Tibiae were first fixed in 10% neutral formalin at 4° C for 15 hr, decalcified in equal volumes of 2% formic acid and 20% sodium citrate at pH 4.9 for not more than 5 days and then washed in distilled water before carrying out the embedding schedules. The celloidin-impregnated tibiae were placed in 70% ethanol to harden the celloidin and then washed in distilled water for 1-2 hr. These tibiae and those embedded in gelatine were cast in a gelatine block which was then hardened in 10% neutral formalin at 4° C for 2 hr. Sections of these and unsupported tibiae were cut at 15 μ on a freezing microtome. Decalcified tibiae embedded and blocked in paraffin wax were sectioned at 15 μ on a base sledge microtome. The enzymes were demonstrated using the coupling azo dye method given by Pearse (Histochemistry, 1st Ed. 1954). The stable diazotates of 4 benzoyl amino 2-5 diethoxyanilene, 3 nitro toluidine and o-dianisidine were used. Of the embedding procedures paraffin wax embedding produced the greatest loss of both enzymes. Gelatine embedding and infiltration with celloidin were equally good for the demonstration of acid phosphatase but for alkaline phosphatase the celloidin method was superior. The gelatine embedded material did not produce consistently good results. Celloidin-impregnated tibiae could be stored without marked deterioration of the enzyme content for longer than gelatine-embedded tibiae.     

Spectrally resolved microscopy of GFP trafficking.

Folding and chromophore cyclization-oxidation processes of green and cyan fluorescent fusion proteins (GFP and CFP) in subcellular microenvironments of transfected C6 glioma cells were studied by multipixel spectrally resolved microscopy (SRM). Discrete time-dependent spectral transitions were characterized during protein folding and chromophore maturation in the cytosol, nucleus, mitochondria, endoplasmic reticulum (ER), and Golgi. Spectral similarity mapping of fluorophore transition phases demarcated spatio-temporal fluorescence correlation at a subcellular level. Folding stages were characterized by a transition from red-shifted spectral populations in the time interval of 7-10 hr after transfection to a fully matured fluorophore emitting typical GFP or CFP fluorescence after 10-15 hr. The nascent protein revealed an initial focal accumulation in cytosol emitting in the range of 580-680 nm. After 10 hr, mixed pixel population spectra were measured and at 15 hr GFP was visualized in the cytoplasm by its specific spectral fingerprints with maxima at 545 nm. For nucleus- and mitochondrion-targeted CFPs, the mature conformer was discovered only in its final destination, whereas intermediate steps of fluorophore synthesis (at 10 hr) were found in the cytoplasm. Enhanced fluorescence maturation was manifested only by the ER-Golgi-targeted CFP after 10 hr post transfection by spectral imaging. Moreover, only remnants of initial intermediate fluorescent pixels were localized externally to the Golgi framework at 15 hr. SRM assessed the competence of ER-Golgi to maintain efficient CFP folding in comparison to the rest of the cellular compartments.     

Fusion of oil bodies in endosperm of oat grains

Few microscopical studies have been made on lipid storage in oat grains, with variable results as to the extent of lipid accumulation in the starchy endosperm. Grains of medium- and high-lipid oat (Avena sativa L.) were studied at two developmental stages and at maturity, by light microscopy using different staining methods, and by scanning and transmission electron microscopy. Discrete oil bodies occurred in the aleurone layer, scutellum and embryo. In contrast, oil bodies in the starchy endosperm often had diffuse boundaries and fused with each other and with protein vacuoles during grain development, forming a continuous oil matrix between the protein and starch components. The different microscopical methods were confirmative to each other regarding the coalescence of oil bodies, a phenomenon probably correlated with the reduced amount of oil-body associated proteins in the endosperm. This was supported experimentally by SDS-PAGE separation of oil-body proteins and immunoblotting and immunolocalization with antibodies against a 16 kD oil-body protein. Much more oil-body proteins per amount of oil occurred in the embryo and scutellum than in the endosperm. Immunolocalization of 14 and 16 kD oil-body associated proteins on sectioned grains resulted in more heavy labeling of the embryo, scutellum and aleurone layer than the rest of the endosperm. Observations on the appearance of oil bodies at an early stage of development pertain to the prevailing hypotheses of oil-body biogenesis.     

Nuclear size and DNA content of the embryo and endosperm during their initial stages of development in Glycine max (Fabaceae)

A technique was developed for isolating embryo sacs from ovules of soybean and for separating embryo from endosperm. Image analysis and cytophotometry were used to determine the relative mass of DNA and size of nuclei of endosperm and embryo cells. Analyses were done at the globular through late heart-shaped embryo stages to correlate ploidy level or nuclear size, and differentiation in these tissues. Mean size of embryo nuclei was fairly constant through all stages studied. Ploidy condition of the embryo was stable, 95%–99% of the nuclei were distributed in a bipolar pattern by relative mass at 2C and 4C. Few embryo nuclei (3%) had ploidy levels above 4C at the late heart-shaped embryo stage. Variability in size of endosperm nuclei seemed correlated with the morphological state of these nuclei (free-nuclear vs. cellular). Most endosperm cells did not show significant polyploidy with 84%–92% of nuclei in the expected 3C–6C range, but some nuclei with elevated ploidy levels were noted during endosperm cellularization. Endosperm senescence was correlated with nuclear DNA loss over time. Polyploidy seems to have no direct role in the early differentiation of the soybean embryo and endosperm, but these stable conditions may be necessary for the early establishment of the embryo.     

Plastic sectioning for light microscopy of Pyrenomycetes.

In preparation for light microscopy, ascocarps of Sordaria fimicola Ces. & DeNot. were embedded in Spurr's medium and sectioned at 1-1.5 mum on an ultramicrotome. Sections were floated on Giemsa staining solution at 60 C for 10-30 min, washed in distilled water, affixed to slides by drying, and mounted in immersion oil. Best preservation of the delicate sterile tissues of the centrum was obtained by fixation in 1% KMnO4 for 2.5-3 hr, followed by the Giemsa stain. This method is suggested for future studies on the morphology of perithecial ascomycetes.     

A Copper Sulfate-Silver Nitrate Method for Nerve Fibers of Planarians

For staining in toto, planarians are fixed in a mixture of 10 ml of commercial formalin, 45 ml of 95% ethanol and 2 ml of glacial acetic acid. After treatment with 70% ethanol 3-10 days, they are washed in distilled water and immersed in 10% CuSO₄. 5H₂O for 3 hr at 50° C, transferred without washing to 1% AgNO₃ for 1.0-1.5 hr at 50° C; and then developed in: 10 ml of 1% pyrogallol, 100 ml of 56% ethanol and 1 ml of 0.2% nitric acid. Gold toning, 5% Na₂S₂O₃ and dehydration follow as usual. For staining sections, material is fixed in the same fixative, embedded in paraffin and sectioned at 10 μ. After bringing sections to water, they are immersed in 20% CuSO₄. 5H₂O for 48 hr at 37° C; then rinsed briefly in distilled water and placed in 7% AgNO₃ for 24 hr at 37° C. They are washed briefly in distilled water and reduced in: hydroquincne, 1 gm; Na₂SO₃, 5 gm and distilled water 100 ml. Gold toning, followed by 5% Na₂S₂O₃ and dehydration completes the process. Any counterstaining may follow.     

Incorporation of radioactive glucosamine into macromolecules at nerve endings

Mice were injected intracerebrally with [¹⁴C]glucosamine, and incorporation into macromolecules in various subcellular fractions of brain was studied at a number of times after administration of the precursor. The [¹⁴C]glucosamine was rapidly incorporated into macromolecules of all the subcellular fractions of brain including both the soluble and particulate fractions of isolated nerve endings. Incorporation into macromolecules in the soluble fraction of nerve endings was quite extensive 3 hr after administration of the precursor and the specific acitvity of this fraction fell thereafter. In contrast there was only slight incorporation of [¹⁴C] leucine into the soluble protein from isolated nerve endings in the first few hours after administration, whereas the other subcellular fractions were maximally labelled at that time. The data suggests that, unlike protein which is largely transported to nerve endings in the axoplasm, there is extensive incorporation of carbohydrate into macromolecules in nerve endings. Whereas the protein component of a glycoprotein or mucopolysaccharide may be transported to the nerve ending from the perikaryon, the structure and function of this protein may be modified at the nerve ending by further incorporation of glucosamine, sialic acid and possibly other carbohydrates. The carbohydrate-containing macromolecules could influence nerve ending function immediately after these final synthetic reactions since these reactions occur at the nerve ending and not in the perikaryon.     

Demonstration of Cholinesterase in Teeth

Human teeth have been studied by treatment with copper thio-choline, the method developed by Koelle for demonstrating activity of both specific and nonspecific cholinesterases. Freshly extracted teeth were collected and immediately sectioned on a cutting machine designed for calcified tissues. One series of teeth was sectioned sufficiently thin for microscopic study. Another series of teeth was bisected to expose the pulp chambers to the reagents. These teeth were divided into 5 experimental groups. The first group was treated with 10^-6M di-isopropylfluorophosphate (DFP) for 30 min at 37°C and then incubated with acetylthiocholine (AThCh) for 16 to 20 hr at 37°C in order to reveal the sites of activity of the specific enzyme, AChEase. The second group was incubated in a substrate of butyrylthiocholine (BuThCh) for 12 to 16 hr at 37°C to indicate the sites of the nonspecific ChEase. The third group was incubated in AThCh for 16 to 20 hr at 37°C without previous treatment by an inhibitor in order to reveal the presence and location of both the specific and nonspecific ChEase. The fourth and fifth groups were utilized as controls. Group 4 tissues were incubated without a substrate while those of group 5 were treated with DFP and then incubated with BuThCh. The specimens then were treated with ammonium sulfide to outline the sites of ChEase activity. The thin sections were mounted directly but the series of halved teeth were decalcified, embedded in paraffin, sectioned and then mounted. By these methods the presence and location of both specific and nonspecific ChEase activity were observed in human teeth. Concentration of specific ChEase was observed along the coronal aspect of the pulp chamber and along the course of the pulpal nerves. The nonspecific ChEase was observed throughout the pulpal tissue and appeared to be concentrated along the nerves and blood vessels. Neither series of control tissues exhibited any staining in the pulp tissue.     

In vitro metabolism of [14C]4-chlorobiphenyl and [14C]2,2',5,5'-tetrachlorobiphenyl by hepatic microsomes from rats and pigeons. Evidence against an obligatory arene oxide in aromatic hydroxylation reactions.

1. The catalytic activities of cytochromes P-450IA1 and P-450IIB1 in control and Aroclor 1254 treated rats and pigeons (1 mmol/kg) were assessed using [14C]4-chloro- and [14C]2,2',5,5'-tetrachlorobiphenyl as substrates. Treatment of rats resulted in increases of the total amount of chloroform-extractable metabolites of [14C]4-chlorobiphenyl from 37.2 (control) to 199.4 and 221.6 nmol/hr per mg microsomal protein at 48 and 120 hr post treatment. The portion of [14C]4-chloro-3',4'-dihydroxybiphenyl (M4) and of a second unidentified dihydroxylated metabolite (M3) increased during these incubations from 13.7% for controls to 53.5% at 48 hr and 69.12% at 120 hr post treatment. 2. [14C]4-chloro-3'-hydroxybiphenyl (M1) and [14C]4-chloro-4'-hydroxybiphenyl (M2) were the major metabolites formed by pigeon hepatic microsomes; however, the amounts formed were 38.7- and 29.3-fold less, respectively, than in untreated rats. Treatment of pigeons with Aroclor 1254 increased the metabolite formation from 1.0 (control) to 13.6 and 22.4 nmol/hr per mg microsomal protein at 48 hr and 120 hr post treatment respectively; however, only small amounts of metabolites M3 (0.5 nmol/hr per mg protein) and M4 (2.0 nmol/hr per mg protein) were detected. 3. Treatment of rats with Aroclor 1254 resulted in an approximately two-fold increase in the rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl, and the ratio of 3- to 4-hydroxylation increased from 0.45 (control) to 0.6 and 0.8 at 48 hr and 120 hr post treatment respectively. The rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl by control and Aroclor 1254 treated pigeons was up to 23-fold lower than in rats and there was no evidence for the formation of the diol metabolite M3. However, as with rats, the ratio of meta- to para-carbon atom hydroxylation increased from 0.58 (controls) to 0.72 at 120 hr post treatment. 4. From the evidence presented, it is suggested that cytochromes P-450IA1 and P-450IIB1 may not metabolize PCB-congeneric substrates via an obligatory arene oxide intermediate.     

Genetic analysis of opaque2 modifier loci in quality protein maize

Quality protein maize (QPM) was created by selecting genetic modifiers that convert the starchy endosperm of an opaque2 (o2) mutant to a hard, vitreous phenotype. Genetic analysis has shown that there are multiple, unlinked o2 modifiers (Opm), but their identity and mode of action are unknown. Using two independently developed QPM lines, we mapped several major Opm QTLs to chromosomes 1, 7 and 9. A microarray hybridization performed with RNA obtained from true breeding o2 progeny with vitreous and opaque kernel phenotypes identified a small group of differentially expressed genes, some of which map at or near the Opm QTLs. Several of the genes are associated with ethylene and ABA signaling and suggest a potential linkage of o2 endosperm modification with programmed cell death.     

Plastic Sectioning for Light Microscopy of Pyrenomycetes

In preparation for light microscopy, ascocarps of Sordaria fimicola Ces. & DeNot. were embedded in Spurr's medium and sectioned at 1-1.5 μm on an ultramicrotome. Sections were floated on Giemsa staining solution at 60 C for 10-30 min, washed in distilled water, affixed to slides by drying, and mounted in immersion oil. Best preservation of the delicate sterile tissues of the centrum was obtained by fixation in 1% KMnO₄ for 2.5-3 hr, followed by the Giemsa stain. This method is suggested for future studies on the morphology of perithecial ascomycetes.     

Assimilation of glucose carbon in subcellular rat brain particles in vivo and the problems of axoplasmic flow

1. Rats were injected with [U-¹⁴C]glucose and the content of ¹⁴C in proteins and lipids of the cerebral P₁ (`nuclear'), P<sub>2</sub> (`mitochondrial'), P₃ (`microsomal') and high-speed supernatant fractions was measured 7, 22 and 93hr. after injection of labelled glucose. 2. The crude brain mitochondrial fractions (P<sub>2</sub>) were subfractionated on continuous sucrose gradients (0·32–1·8m-sucrose) and the <sup>14</sup>C content of the proteins and lipids of about 20 subfractions was measured. 3. About 40–50% of the <sup>14</sup>C assimilated by brain proteins was found in the P<sub>2</sub> (`mitochondrial') fraction. About 68–70% of the ¹⁴C assimilated by brain lipids was also recovered from the lipids of the P₂ fraction. 4. Between 22 and 93hr. after injection of [U-¹⁴C]glucose both the amount of ¹⁴C in the protein of the P₂ (`mitochondrial') fraction and the specific activity of this protein increased. The specific activity of the protein of all other particulate fractions (P₁, P₂ and P₃) and subfractions (obtained from sucrose-density-gradient subfractionation of fraction P₂) when related to the specific activity of the high-speed supernatant protein also increased during 93hr. after injection of [U-¹⁴C]glucose. The amount of ¹⁴C in the protein of the high-speed supernatant and the specific activity of this protein decreased during the same period. 5. The distribution of ¹⁴C in the lipids of all subcellular particulate fractions remained unchanged during the period 22–93hr. after injection of [U-¹⁴C]glucose. 6. It was concluded that a diffusion occurs of some supernatant proteins into subcellular particulate matter of the cerebrum and no significant preference for any subcellular particulate matter was observed. The lipids occur in the cerebrum mainly in a non-diffusible state, which is consistent with the view that they form almost entirely a part of the structure of the cerebrum. 7. The data obtained do not lend further support to the concept of axoplasmic flow within the cerebrum or the concept of a one-directional flow of mitochondria or other subcellular particles within the cerebrum.     

Rapid bleach for melanin

Complete bleaching of melanin in strongly pigmented specimens embedded in paraffin or polystyrene, and sectioned and mounted on slides, is possible in 1-3 hr at 37 C in a solution of 20 ml of benzyl alcohol, 10 ml of acetone, 5 ml of 10% hydrogen peroxide and 4 drops of a 25% ammonia solution. The bleached tissues are well preserved and tolerate further histochemical treatments. All the stains and reactions tested give results identical to or better than those obtained after 24-48 hr oxidation in 10% hydrogen peroxide.     

Expression and localization of human lysozyme in the endosperm of transgenic rice

In order to understand the characteristics of recombinant protein expression and sublocalization in rice ( Oryza sativa L.) endosperm, we examined the expression level of human lysozyme protein and its subcellular location in transgenic rice seeds driven by rice glutelin and globulin promoters and signal peptides. A time course of human lysozyme expression during endosperm development was analyzed. The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins. Immunofluorescence microscopy revealed that human lysozyme and storage proteins co-localized to type-II protein bodies. Both promoter-signal peptide parings targeted recombinant protein to the protein bodies. In addition, a transgenic line with a higher lysozyme expression level exhibited morphologically different protein bodies with an unbalanced composition of lysozyme and native storage proteins. The high-level expression of recombinant protein distorted the trafficking and sorting of native storage proteins in rice endosperm and affected the expression of native storage protein.     

Serial Sectioning of Insects with Hard Exoskeleton by Dissolution of the Exocuticle

The method reported here was designed to produce paraffin serial sections as thin as 5 Mm of insects or other arthropods with a hard cuticle. Heads and abdomens of Apis mellifera, Eristalomyia tenax and Tenebrio molitor were fixed with Schaffer's liquid, dehydrated with 80% ethanol, 90% ethanol, two changes of 100% isopropanol (2 hr each) and 12 hr in a 1:1 mixture of paraffin (58 C melting point) at 60 C. They were molded in paraffin after 12 hr of infiltration under a partial vacuum at 60 C. Large body openings of objects were sealed with paraffin to prevent infiltration of solvents.

Thereafter, the outer paraffin was removed manually and with xylene (15 min); the cuticle was rehydrated with 100% isopropanol and 100% ethanol (15 min each). The objects were then treated with Sputofluol (Merck; a mixture of NaOH and NaCIO) until they became white or their colorless endocuticle was stainable with aniline blue WS (C.I. 42755) after rinsing in a 50% acetic acid solution (v/v). They were then dehydrated with 100% ethanol and 100% isopropanol (15 min each) and subsequently re-embedded in paraffin. They were molded, sectioned, stained and mounted as usual.