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Postfixation with osmium tetroxide and Epon embedding are essential for the preservation and visualization of subcellular ultrastructures via electron microscopy. These chemical treatments diminish the fluorescent intensity of most fluorescent proteins in cells, creating a problem for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded mammalian cultured cells. We found that two green and two far-red fluorescent proteins retain their fluorescence after chemical fixation with glutaraldehyde, osmium tetroxide-staining, dehydration, and polymerization of Epon resins. Consequently, we could observe the fluorescence of fluorescent proteins in ultrathin sections of Epon-embedded cells via fluorescence microscopy, investigate ultrastructures of the cells in the same sections via electron microscopy, and correlate the fluorescent image with the electron microscopic image without chemical or physical distortion of the cells. In other words, referred as "in-resin CLEM" of Epon-embedded samples. This technique also improves the Z-axis resolution of fluorescent images. In this chapter, we introduce the detailed protocol for in-resin CLEM of Epon-embedded mammalian cultured cells using these fluorescent proteins.

Super-resolution correlative light and electron microscopy (SR-CLEM) is a powerful approach for imaging specific molecules at the nanoscale in the context of the cellular ultrastructure. Epon epoxy resin embedding offers advantages for SR-CLEM, including ultrastructural preservation and high quality sectioning. However, Epon embedding eliminates fluorescence from most fluorescent proteins. We describe a photocontrollable fluorescent protein, mEosEM, that can survive Epon embedding after osmium tetroxide (OsO4) treatment for improved SR-CLEM.

Ultrastructural artifacts regarding collapse and aggregation of cultured cells have been problematic, especially when investigated apoptotic cells. The infiltration process during sample preparation is considered to be the most crucial factor for this problem. This study was conducted using two culture systems: a suspension culture system of human T-lymphocyte Jurkat cells and rabbit mature dendritic cells and a monolayer culture system of human lung macrophages, human breast cancer cells (A-546 cells) and cat bone-invasive gingival cancer cells (sccf3 cells). Fixation was conducted prior to removing or detaching the cells from the culture dishes. Initial infiltration with a 1 : 3 volume ratio of epon resin : propylene oxide was found to be the most crucial step among these cultured cells. The improved epon-resin infiltration method could eliminate the artifacts. Thus, differentiation between artifactual images and true images is highly possible.

We studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy.

An immunofluorescence staining method for Epon-embedded materials is described. Rat kidney and liver were fixed by perfusion with 1% glutaraldehyde for 10 min. Tissue slices were embedded in Epon. Semithin sections with thicknesses ranging from 1,000 to 100 nm were cut and mounted on clean glass slides. Epoxy resin was removed by treatment with 10% sodium ethoxide. Sections were digested with 0.05% trypsin and then treated with sodium borohydride. Sections were immunostained for leucine aminopeptidase (plasma membrane), catalase (peroxisomes), 3-ketoacyl-CoA thiolase (mitochondria), cathepsin D (lysosomes), and LGP107 (lysosomal membrane) using Cy(2)- or Alexa 546-labeled secondary antibodies. In 1,000-nm-thick sections, non-specific fluorescence remained and such fluorescence decreased as the sections became thinner. Clear specific fluorescence was obtained in the sections with thicknesses ranging from 250 to 100 nm with Alexa 546-labeled antibody (red fluorescence) but was not specific enough with Cy(2)- or Alexa 430-labeled antibody (green fluorescence). Sodium borohydride greatly abolished autofluorescence of glutaraldehyde. The present method made it possible to obtain signals in cross-sections of biological materials with a thickness of 250-100 nm, which are difficult to obtain in optical section using a confocal laser microscope.

Nanogold is a tiny gold probe, freely diffusible in cells and tissues, and is suitable for pre-embedding immunohistochemistry. However, it is necessary to develop Nanogold to a larger size so that it can be observed by conventional transmission electron microscopy. Silver enhancement is usually used for visualizing Nanogold, but the silver shell produced is unstable in OsO4 and often becomes invisible after OsO4 postfixation, which is necessary for good visualization of ultrastructure. We used silver enhancement with silver acetate, followed by gold toning with chloroauric acid, to replace the silver shell with a more stable gold in order to observe Nanogold after osmium fixation and Epon embedding. This technique is applicable to various intra- and extracellular antigens. For correlative observation of immunolabled specimens by light and electron microscopy, specimens adhered to slideglasses were embedded in Epon under nonadhesive plastic film. By heating the Epon sheets after polymerization, these supports were removed without difficulty and provided easy correlative observation.

We evaluated bromodeoxyuridine (BrdU) immunohistochemistry of paraffin-, methyl methacrylate (MMA)-, and epon-araldite (epon)-embedded canine bone specimens to establish an optimal technique for studying cell kinetics of fracture healing in a canine tibial gap model. Dogs were sacrificed 4 months after tibial ostectomy and 1 hr after i.v. injection of BrdU (100 mg/kg). BrdU immunohistochemical staining with a peroxidase-labeled streptavidin-biotin (LAB-SA) method was performed on thin sections of tibia fixed in 70% ethanol and embedded in paraffin, MMA, or epon. Thin section of small intestines fixed in 70% ethanol and 10% neutral buffered formalin and embedded in paraffin, MMA, or epon were BrdU-stained and served as a model for proliferating tissue. Good and consistent BrdU immunostaining without detachment of bone sections was obtained for epon-embedded undecalcified bone sections. BrdU-positive cells were easily identifiable, in contrast to negligible background staining. BrdU-labeled osteoprogenitor cells and osteoblasts were observed around and on the surface of woven bone in external and internal callus of the ostectomy gap. Nuclei of osteocytes were not labeled. In contrast to the epon-embedded specimens, BrdU immunostaining of paraffin-embedded decalcified and MMA-embedded undecalcified bone specimens was unsatisfactory. The results of this study suggest that BrdU immunohistochemistry of ethanol-fixed, epon-embedded, undecalcified canine bone sections is a technique suitable for study of fracture healing with the described methodology.

A simple procedure is described for removing Epon resin from semi-thin 1 micron sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4-7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwell's solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwell's solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results.

The availability of high-resolution field emission scanning electron microscopes (FESEM) and the recent development of a less destructive method for extracting Epon from sections motivated us to investigate these techniques for the study of internal cell structures. We chose the nuclear pore complex (NPC) and insect striated muscle as test objects. Chemically fixed or rapidly cryoimmobilized samples were embedded in Epon 812. The Epon was extracted from 200- to 300-nm-thick sections with a modified potassium methoxide-crown ether complex. The samples were viewed with high-resolution FESEM at low voltages. In tangential sections of isolated nuclear envelopes from Xenopus oocytes the cytoplasmic and intranuclear components ("fishtraps") of NPCs appeared identical to what has been described from whole mounts. In cross sections, fishtraps are seen in side view, which is possible only with this technique. In longitudinal and cross sections of insect flight muscle the classical arrangement of myofilaments and cross-bridges is well preserved. This method now makes it possible to image internal cell structures from any desired angle by high-resolution FESEM. Immunolabeling studies on the rabbit psoas muscle demonstrated that antigenicity of alpha-actinin was retained in Epon-extracted sections. Immunogold labeling with antibodies against alpha-actinin conjugated to 3-nm gold beads was intense, highly specific, and restricted to the Z lines. This method can overcome the penetration problem of immunogold labeling, since any cell component can be positioned at the surface of the section. Obviously this approach can become a powerful new tool for many areas of structural cell biology.

Human, rat and mouse pituitary tissues have been examined electron microscopically in transmission (TEM), scanning-transmission (STEM) and scanning (SEM) modes for the surface appearance of the secretory granules in tissue sections. Cryofixed and cryosectioned tissue showed only slightly protruding granule profiles which had a smooth surface. Cryofixed, freeze-dried and Epon embedded pituitaries, on the other hand, demonstrated swollen and furrowed surfaces over the granules after contact with water. This topography could also be seen after glutaraldehyde fixation but less after post-fixation in OsO4. The surface alterations in the sections of pituitary secretory granules are thought to be due to differences in the homogeneity of the resin infiltration, leaving resin-free openings where water can enter. It also seems probable that the Epon resin is more influenced by water than has been previously assumed, based on the findings of efficient elimination of osmium from the granules after incubation of tissue sections in water for only 10 min.

In the present study we describe a method for the histochemical demonstration of β-D-galactosidase activity on tissue sections processed for light microscopy at high resolution. 5-Bromo-indolyl-β-D-galactopyranoside (Bluo-Gal) was utilized as an indigogenic method for the demonstration of Escherichia coli β-D-galactosidase reporter gene activity whose expression was studied in a transgenic line where the enzyme, with a nuclear localization signal (nlacZ), is under the transcriptional control of a striated muscle-specific promoter. At the light-microscopic level, by using Differential Interference Contrast (DIC) optics, the reaction product was detected as precipitates in the form of fine birefringent crystals. These were located around and inside the nuclei of β-gal-expressing cells. This simple method allows an easy and rapid identification of few or even one labeled cell(s) within large microscopic fields (whole embryos) and the labeled cell(s) can be evaluated both morphologically and quantitatively.

We describe here a procedure that significantly enhances the intensity and method-specificity of immunocytochemical staining in large mounted semi-thin plastic Epon sections. The procedure was developed for the detection of the neurotransmitter gamma-aminobutyric acid (GABA) in rat brain tissue fixed with glutaraldehyde, but it may also be helpful in unmasking other antigens under different conditions. In addition to some practical suggestions for improving the reproducibility of the staining procedure, we demonstrate that the crucial step in the procedure is pre-treatment of the deplasticized sections with proteinase-K before exposure to the first antibody. This leads to a high morphological resolution and an excellent immunocytochemical signal.

It is desirable to shorten expose time in 3H-autoradiography. We developed a new solid scintillator, a mixture of DPO, POPOP and Epon 812, for whole-body autoradiography. The new scintillator is originally in a sol form, but quickly transforms to a solid homogeneous film following exposure to room air. The new scintillator enabled whole-body autoradiography to be developed faster when compared with a liquid scintillator or commercially available reagents. We also demonstrated that the new scintillator gave consistently high counting performance and thus shortened duration of exposure when used in whole-body autoradiography, whereas commercially available enhancers were found to be unsuitable for autoradiography, resulting in dappled images. Furthermore the quantitative analyses of the autoradiograms by a densitometer demonstrated the highest density, and ratio with this new solid scintillator among those with a liquid scintillator and commercially available reagents. In conclusion, a new solid scintillator, a mixture of DPO, POPOP and Epon 812, is a useful and cost effective enhancer for the whole-body autoradiography of tritum compounds.

A simple and rapid method for fixation, processing, and Epon embedding of cells grown on a filter support is described. The results show that it is possible to embed filter-grown cells in resin within 30 min of applying the primary fixative and still to achieve good ultrastructural preservation.

The use of a conjugate of colloidal gold with monotypic antibodies against a neutral Mn(2+)-dependent DNAse has revealed the enzyme localization in ultrathin Epon sections of glutaraldehyde fixed tissues. Technical procedures involved in fixation, embedding, and immune reactions are described. DNAse has been established in the nuclei in both normal and regenerating liver. A protein, immunologically close to DNAse, has been identified in the nuclei of a lymph node, thymus, spleen and cerebellar cortex.

We evaluated bromodeoxyuridine (BrdU) immunohistochemistry in undecal-cified adult rat tibiae to study cell kinetics in various bone compartments: primary and secondary spongiosae, periosteum, and bone marrow. Several regimens of BrdU administration were tested (IP injections and osmotic minipumps). We compared LR White resin, methylmethacrylate, and Epon–araldite embedding, microwave irradiation for antigen retrieval, several concentrations of sodium ethoxide for deplastification, and various DNA de-naturation procedures. Paraffin-embedded decalcified tibiae and Epon-embedded bowel were used as positive controls. The best results were obtained in rats labeled with 40 mg of BrdU for 72 hr using osmotic minipumps. The procedure using a Microprobe system in Epon-embedded bone tissue with a sodium ethoxide concentration of 50% for two intervals of 20 min provided the best staining quality and tissue preservation. Labeled pre-osteoblastic cells and bone marrow cells could be counted. Epon embedding allowed preservation of tetracycline double labeling performed 1 to 5 days before sacrifice. The number of labeled pre-osteoblastic cells was correlated with the double-labeled surface area measured histomorphometrically.

Immunocytochemistry for amino acids with post-embedding gold is compatible with glutaraldehyde fixation, osmication, and embedding in epoxy-based plastics, but immunogold detection of larger molecules in the central nervous system commonly requires special procedures, e.g. minimizing exposure to glutaraldehyde, eliminating osmium, cryosectioning, and/or embedding in acrylic plastics. These make samples more difficult to prepare and view and may compromise structural preservation. We report a new technique, fixing with high levels of glutaraldehyde, replacing osmium with tannic acid followed by other heavy metals and p-phenylenediamine, and embedding in Epon. This method optimizes antigenicity while retaining the structural preservation and convenient handling of standard embedding techniques. Compared to standard Epon embedment, labeling for neuropeptides in brain and spinal cord is improved. Moreover, the present method yields excellent labeling of glutamate receptors (difficult to identify with traditional post-embedding techniques) and enables simultaneous visualization of associated neurotransmitters.

Siderosomes (i.e. single membrane-bound lysosomal bodies containing haemosiderin) were produced in the liver and muscle of rats by injections of iron dextran. Electron-probe X-ray analysis was executed on siderosomes in cryosections of quick-frozen fresh unfixed tissues (liver and muscle) and sections of Epon-embedded tissues. There were no statistically significant differences between the ratios of iron:phosphorus and iron:sulphur in these two types of preparations. Hence it is concluded that there is no significant loss or gain of phosphorus or sulphur during preparation of tissues for Epon embedding. The results confirm past belief that so little phosphorus or sulphur is present in siderosomes that haemosiderin is best regarded as ferric hydroxide oxide. A new finding in the present study was the demonstration of small amounts of potassium in siderosomes in cryosections. It seems that potassium is lost (leaches out) from siderosomes during preparation of tissues for Epon-embedding.

A reliable, sensitive, high-resolution method with good structural visualization for preembedding immunoelectron microscopy was proposed. The present technique involves the following processes. Immunolabeling of cryostat sections with primary antibodies and with nanogolds, silver intensification for visualization of nanogold secondary antibodies, gold-toning for stabilization of silver shells, and osmium postfixation and embedding in Epon for good structural visualization. The technique was applied to rat testis with anti-laminin and anti-fibronectin antibodies, showing that these two proteins are localized differentially in the lamina propria of the tissue.

Epon-embedded biological materials exhibit well preserved ultrastructure but generally weak antigenicity. Brief etching of ultrathin Epon sections with resin solvent, ethanolic sodium hydroxide, brought about a nearly 2-fold increase in the immunogold labeling density of rat and human hepatic peroxisomes after incubation with antisera against catalase and 3 enzymes of lipid beta-oxidation: acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase. The etching was superior to pretreatment with an oxidant, sodium metaperiodate. Despite some deterioration of the cellular ultrastructure, the obtained enhancement of the sensitivity of the protein A-gold method may be helpful in cases when the antigenicity to be detected is strongly inhibited by epoxy resin embedding.

The efficiency of various postembedding procedures for actin immunogold detection was compared using testicular tissue as a model. Whatever the fixative, testes embedded in LR White resin or in Lowicryl K4M showed few differences as regard ultrastructural preservation and gave similar actin antigenicity preservation. A purified polyclonal antibody (IgG) and a monoclonal antibody (IgM) visualized with gold secondary antibody yielded high labeling intensity whereas the IgG-protein-A gold association was less efficient. Crude antisera gave a low specific staining/background ratio. Samples of testes, fixed in different conditions, were also embedded in Epon, omitting propylene oxide and lowering polymerization temperature to 40 degrees-50 degrees C. This slight modification improved ultrastructural preservation which was better than with hydrophilic resins, as well as made possible immunogold detection of actin though antigenicity preservation was lesser than with these resins. Thus, in Epon embedded samples actin labeling, using IgG antiactin-gold secondary antibody, was similar to that observed after hydrophilic resin-protein-A gold procedures. In addition to actin labeling of various somatic cells it was confirmed that actin is a consistent component of the subacrosomal space of spermatids during the greater part of spermiogenesis in rat.

Correlative light and electron microscopy on the same cells of the adrenal gland of the frog, Rana nigromaculata, fixed in glutaraldehyde followed by osmium tetroxide, was done using the adjacent Epon embedded sections. Electron microscope observation revealed three different types of granule-filled secretory cells; the noradrenaline-storing cells (NA cells) filled with intensely dense and varying shaped granules, the adrenaline-strong cells (A cells) filled with relatively less dense granules and the summer cells (STILLING, 1898) containing very large, round or polygonal granules (0.2-1.3 mu in diameter). Light microscopically, an essential difference could be observed in the affinity to ammoniacal silver solution between NA and A cells. It was clarified that the granules of NA cells stained in black and were clearly distinguishable from the yellow- or brown-stained granules in both A cells and summer cells. This silver method can be applied for the light microscopic identification of the NA cells in the Epon-embedded sections. Furthermore, after immersing the thick sections in toluidine blue or methylene blue, the granules of NA cells showed much stronger affinity to both dyes than those of A cells and became dark blue and occasionally stained greenish blue in methylene blue, while the summer cells became blue and the granules of the A cells stained light blue.

A quantitative evaluation of spermatogenesis in the guinea pig was made at the light microscope level following improved methods of fixation and plastic embedding. The pattern of germ cell differentiation was studied by counting the average number of germ cell nuclei per Sertoli cell nucleus during the seminiferous epithelial cycle. Appreciable cell loss occurred during spermatogenesis. As a result, only 20.8% of the theoretically possible number of spermatozoa were produced from each spermatogonium. A slightly better yield of germ cells was obtained from Epon sections in comparison to paraffin sections. Epon sections can be effectively used for quantitative analyses of various cell populations in small biopsy specimens of testis tissue.

Summary Experiments were performed aiming at establishing optimal conditions for demonstrating the activity of dehydrogenases in Epon -embedded, semithin sections. Small fragments of the nervous tissue were incubated for 1, 4, 10 and 24 h at 37, 20 and 4 °C in the presence of NBT or TC NBT. The incubated fragments were embedded in Epon and semithin sections prepared thereof. The activities of succinate dehydrogenase and of α-glycerol dehydrogenase was evaluated. The experiments have shown that semithin, Epon -embedded sections may be used for demonstration and cytological localization of respiratory enzymes. Various experimental conditions were tried. Optimal results were obtained when unfixed tissue fragments were incubated for 24 h at 4 °C.

Epon embedding was used with the Falck-Hillarp Technique in a study of rat gastric endocrine cells in order to improve morphological resolution. The epon embedding not only produced excellent morphological results, but also resulted in the long-term preservation, now for 10 years of the serotonin-associated fluorescence in the gastric endocrine and mast cells.

SUMMARY Dark gold sections of osmium tetroxide-fixed, Epon-embedded brown adipose tissue before and after low-temperature oxygen plasma microincineration were examined using a high-resolution scanning transmission electron microscope and an energy-dispersive X-ray spectrometer. Microincineration produced ash patterns which were free of organic matrix, chlorine (from the Epon) and probably osmium (from the fixative). X-ray sensitivity was improved by a factor of 2–4 owing to decreased background, and sulphur, calcium and probably phosphorus were detected in the ash. Fidelity of the ash patterns permitted microanalytical spatial resolution of 0.1 μm or better. Oxygen plasma microincineration is thus shown to offer advantages for high resolution X-ray microanalysis of conventionally sectioned biological material. Its future application to shock-frozen, frozen-dried, unstained sections is indicated.

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO4 fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H2O2, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic fibers. Intracytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.

Four-week-old Holtzman rats were injected intraperitoneally with 20 μCi 125I. Six or eight weeks later, they were killed by intracardiac perfusion with glutaraldehyde; thyroid and adrenal glands were excised, postfixed in osmic acid, and embedded in Epon. Steps in the staining procedure of 0.5-1 μm thick sections are: oxidation in 0.3% potassium permanganate in 0.625% sulfuric acid, 2-5 min at 70 C; brief rinse; bleaching with 2.5% NaHSO3, 4-5 min; brief r-utse; let dry completely; aldehydefuchsm, 15-20 min at 50 C; 95% alcohol; rinse in absolute alcohol; let dry completely. seaions were coated with Kodak NTB2 emulsion and exposed for 3 to 8 weeks. Results indicate that (1) tissues are well stained even after an 8-week exposure, (2) aldehyde-fuchsin pduces no chemographic effect, and (3) structures underneath the emulsion are easily identified.

Summary The dinosaur bone was examined with the aim to detect in it the presence of mucopolysaccharides. The methacrylate- and Epon-embedded semithin sections of the studied bone were found to contain substances giving positive reaction on application of histochemical procedures used for identification of mucopolysaccharides (mucosubstances). These substances were also shown to be localized in the perivascular space, within the vascular canal, and around the osteocytes. The distribution of mucopolysaccharides in the dinosaur bone turned out to be comparable with their distribution in the bones of contemporary animals.

SUMMARY This paper describes a method for demonstrating amyloid in human tissue embedded in epon using a combination of congo red staining and polarizing microscopy. The method involves the removal of the embedding medium from semi-thin sections prior to staining with congo red and examination in the polarizing microscope. Typical apple green birefringence was detected in areas of amyloid deposition; these areas were mapped for selective trimming and ultra-thin sections taken serially were examined in the electron microscope. The method was found to be reliable, easy to use and particularly of value as a sampling procedure prior to investigation in the electron microscope.

Summary The histochemical demonstration of polyanionic mucopolysaccharides (glycosaminoglycans) by Alcian Blue at different pH values was applied to 0.5 to 1.0 μm sections of Epon-embedded cartilage and salivary gland. The intact sections did not bind the stain when stained up to 3 h. After partial removal of the resin with NaOH/absolute ethanol solution a selective staining of the cartilage intercellular matrix as well as the salivary gland secretory granules was observed. The optimal time of the resin-solvent action was 2 to 5 min and the staining obtained this way was abolished by the presence of 1 M MgCl2 in the staining solution and by preincubation of sections in 10−2 M cetyl pyridinium chloride [CPC]. When sections were kept in NaOH/ethanol for 30 min, a stronger, diffuse staining of most tissue structures occurred, which was not inhibited by preincubation of sections in CPC even for as long, as 18 h, indicating a loss of the staining specifity.

Semithin sections from glutaraldehyde-fixed, Epon-embedded tissues were stained by aqueous solutions of pyronin Y at increasing concentrations (from 10(-6) to 10(-3) mol). Mucopolysaccharide containing structures (e.g. mucin) were found stained in orange, meanwhile the chromatin and remaining tissue components appeared in a bright pink-red color. Cytophotometric measurements showed that a metachromatic shift occurs in the mucin content from goblet cells after pyronin Y staining at 10(-3) mol. Some features of the metachromatic reactions by cationic dyes are briefly discussed.

SUMMARY The internal fine structure of extraocular muscle cells was observed with scanning electron microscopy. One micrometre Epon sections were etched with an iodine acetone solution. When adhered to carbon coated glass, such specimens may be viewed without surface coating. Such preparations provide information about the fibrillar organization of the muscle fibre, as well as its transverse tubular system and M line.

Methods for light and electron microscopic comparison of individual argentaffin and argyrophil enterochromaffin cells (EC) in the sheep duodenal mucosa are described. These silver procedures were applied for light microscopy to Epon-embedded sections. The adjacent sections were examined with the electron microscope. The most specific characteristics of the argentaffin and argyrophil EC in electron microscopy are highly osmiophilic cytoplasmic granules. In one cell type these granules are smaller and more roundish than in the another type. These two cell types are stainable both by the argentaffin and argyrophil reactions. No essential difference can be observed in the localization of these elements. It is suggested that both cell types belong to the enterochromaffin system. Both silver methods are also suitable for the light microscopic identification of other intestinal structures in sections adjacent to that sectioned for electron microscopy.

A convenient method is described for the removal of Epon 812 from thin sections, utilizing a saturated solutions of sodium hydroxide. The tissue architecture and ultrastructural details are preserved. Hematoxylin-eosin, periodic acid-Schiff and phosphotungstic-hematoxylin staining modifications are suggested which result in differentiation similar to that seen in paraffin sections. The technique is applicable to ultrathin sections suitable for examination with the electron microscope, allowing comparison of staining characteristics and fine structure of adjacent thin sections.

Sixteen-week-old dogs were inoculated with 20,000 tissue culture doses (TCD50) of infectious canine hepatitis virus. Four days after inoculation, liver cells examined in the electron microscope contained both virus particles and nonviral paracrystalline formations. Tissue culture cells also contained virus particles and nonviral paracrystalline formations 48 hr after inoculation with virus. When Epon-embedded sections were treated for 1 hr with 0.5% pepsin in 0.1 N HCl, a selective extraction of the viral coats and of the nonviral paracrystals was observed.

Tissue blocks or sections immunofluorescent stained before embedding, i.g., liver and kidney, can be stored for more than 3 years without demonstrable fluorescence decay. The processing steps, including poststaining dehydration by alcohols and embedding in expoxy resins, seem to stabilize the fluorochromes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) so that they fade less during illumination. This is an advantage of the pre-embedding, immunofluorescent staining technique which is combined with a lack of damage to the antigens by the plastic embedding medium.

The autofluorescence of tissue components in Epon semithin sections depends on the exciting wavelength, section thickness, and the structure under observation. The degree of autofluorescence emission of a given component affects the fluorescence reaction induced by fluorochromes, a feature which must be taken into account when fluorescence microscopy is applied to the study of Epon sections.

Different results of PAS reaction performed on semithin sections of Epon-embedded tissues were obtained when the sections were exposed for various time intervals to saturated solution of NaOH in absolute ethanol, the reagent known to dissolve the resin and facilitate staining. In the untreated sections only glycogen revealed strong PAS reaction, most mucosubstances reacted weakly or not at all. After 1 to 5 min of NaOH in absolute ethanol treatment results observed were in accordance with those usually seen in paraffin sections. When, however, the time of Epon-dissolving procedure was longer than 10 min, most tissue structures demonstrated positive, largely diffuse staining. The mechanism of the observed effect and its significance for the application of PAS reaction to Epon sections are discussed.

The construction and operation of a simple device for serially mounting small Epon sections for light microscopy are described. This device allows the middle portion of the slide to serve both as a shallow reservoir behind the knife edge for flotation of serially oriented sections, and as a miniature hot plate for the subsequent flattening and adhesion of the sections. The advantages of this device, which gave consistently good results with Epon embedded Argas (Persicargas) arboreus tick tissues, are discussed.

Thick sections of tissue, (20 micron--60 micron), are useful in studying the relationship between individual large cells and cell layers in organized neural structures. The ability of the Nomarski Differential Interference-Contrast Microscope to bring a single thin layer into sharp focus makes the examination of such sections feasible. Although celloidin is the classical embedding medium for large, thick sections of neural tissue, the time necessary for this preparation is most inconvenient. Epon is an excellent embedding medium; however, it is extremely hard and brittle. By heating the Epon block face, thick sections can be cut. To avoid the cumbersome, often detrimental use of heat, a modification of this technique was found. Epon blocks, trimmed to a 1 millimeter square face, may be sectioned at room temperature on the sliding microtome at 20 micron to 60 micron with ease. The simple method of preparing such sections is described.

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 hr in 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.

A method for the application of Heidenhain's iron hematoxylin staining to epon-embedded tissue sections is described.Various tissues of the mouse were fixed with glutaraldehyde followed by osmium tetroxide, embedded in epon-resin, and cut into semi-thin sections about 1μ thick. The sections were affixed to the slide surface by heating on a hot-plate.Heidenhain's iron hematoxylin solution was prepared according to ROMEIS' recipe. The staining procedure which has been utilized for paraffin and celloidin sections was applied to the epon-sections with slight modification, e. g., heating the utilized solutions. The embedding medium was not removed for the staining.The results of the present staining show that the connective tissue fibers, nuclear chromatins and nucleoli were commonly stained in dark shades of black, while the cytoplasm and nuclear sap showed light shades of yellowish brown. The keratinized layer of stratified squamous epithelium, glycogen areas of hepatic cell, goblet cell mucus, Paneth cell granules and the striated border of intestinal epithelium were selectively emphasized in dark shades. On the other hand, the sarcoplasm of muscle fibers, myelin sheaths and erythrocytes appeared in light shades. Under mounting with Canada balsam the stain remains stable for at least a year.The present method is useful especially in tissues containing a cosiderable amount of connective tissue fibers but is valid also as a general stain for all epon-embedded tissues.

Tissue which had been fixed in 4% glutaraldehyde and postfixed in 2% OsO4 was subsequently treated with p-phenylenediamine, either in the block prior to embedding in paraffin or Epon or, in the case of Epon-embedded material, after sectioning for light microscopy. The p-phenylenediamine was best used as a 0.8-1% solution in 70% alcohol. The p-phenylenediamine caused a very considerable intensification of staining of any cell components; this intensification of staining was particularly marked in the case of the lipid granules of renal medulla.

Summary Phosphotungstic acid (PTA) binds to the basic dye pyronin Y to give a non fluorescent complex with absorption characteristics which are different from those of the free dye. The use of this complex on different cells and Epon embedded tissues revealed no staining affinity and only certain hydrated tissue components as starch showed a light reaction with the pyronin-PTA. The role played by the hydratation degree of certain components from epoxy-embedded tissues on the reaction mechanisms observed with some dyes and electron contrasting agents is briefly discussed.

Procedures for ultrastructural postembedment immunolocalization of antigens were investigated by use of the indirect peroxidase labelled antibody method. Results were compared with those obtained with the ultrastructural preembedment technique. IgG globulins in IgG synthesizing cells of rat lymph nodes served as model. Formaldehyde fixation, ethanol dehydration and embedment in Epon 812 did not abolish immunoreactivity. Sufficient numbers of antigens remained for subsequent postembedment immunohistology. Antigen binding sites were readily localized in ultrathin sections. For this purpose, polymerized resin had to be partially removed. Sodium methoxide in methanol/benzene mixture rapidly dissolved ultrathin sections. Diluted alcoholic sodium hydroxide enabled reliable resin etching and subsequent immunostaining. Treatment of ultrathin sections with hydrogen peroxide alone, did not permit immunolocalization of IgG. Optimal antigen detection was attained with antibodies isolated by affinity chromatography and purified peroxidase conjugates. IgG was stained in the rough-surfaced endoplasmic reticulum, the perinuclear space and the Golgi apparatus; the subcellular distribution corresponded to that obtained with preembedment immunohistology. In the latter technique, substrate accumulation was more homogenous than in postembedment staining.

To study cytological changes in the migrating cell layer of the ameloblasts in the rat incisor with semithin Epon sections the ideal section plane would consequently be along the midsagittal plane of the incisor. However, the small dimensions and the complex three-dimensional structure of the apical part of the odontogenic organ covered by alveolar bone make it difficult to orientate and position sections in the midsagittal plane. Serial sectioning of the apical half of the maxillary incisor in the horizontal and sagittal planes clarified the relation of the midsagittal plane of the odontogenic organ to the alveolar bone, and the changes of the sectional profile of the odontogenic organ as a function of its position on the transverse axis of the incisor. From this information a method was designed whereby the two axes of the midsagittal plane were marked directly on the block surface before sectioning, and the position of sections in the midsagittal zone of the ameloblasts standardized.