Postembedding immunogold labeling for electron microscopy using "LR White" resin

A method is described for performing postembedding immunogold immunocytochemistry on sections of LR White-embedded tissues. Fixation of tissue in a combination of paraformaldehyde and glutaraldehyde, or with low concentrations of glutaraldehyde followed by partial dehydration, resulted in preservation of antigenicity for a variety of proteins in different tissue samples. Good structural preservation facilitated high-resolution immunolabeling when coupled with the use of purified monoclonal antibodies. The technique is straightforward and versatile, offering the potential for many immunocytochemical applications with minimal modifications.     

The morphological and immunohistochemical analysis of renal biopsies by light and electron microscopy using a single processing method

A methodology is described in which a number of well-established research techniques are brought together to enable the complete diagnostic analysis of a renal biopsy on a single piece of tissue. By embedding the biopsy in the acrylic resin LR White, unsupported sections of which are stable in the electron beam, light and electron microscopy and immunocytochemistry become feasible on sections from the same block. The biopsy is glutaraldehyde fixed but post-fixation in osmium tetroxide, which is often deleterious to antigen preservation, is omitted. Extraction in organic solvents and resin monomer is minimized by rapidly infiltrating the tissue from 70% ethanol and polymerizing the resin catalytically at 0 degrees C. Semithin sections can be stained with haematoxylin and eosin, Toluidine Blue or methenamine silver, giving results similar or superior to those obtained from paraffin sections. Thin sections show that the standard of morphological preservation is similar to that seen using epoxide sections even though the kidney is unosmicated. The tissue retains a high level of antigen reactivity, which, in the limited number of cases so far examined, has paralleled or exceeded that demonstrated by conventional immunofluorescence on frozen sections.     

Light and electron microscopic immunocytochemistry of the same nerves from whole mount preparations

A technique for performing correlated light and electron microscopic immunocytochemical studies on whole mount preparations has been developed using myenteric plexus from guinea pig small intestine as a model. With this method a structure containing a particular antigen can first be located by light microscopy and then examined with the electron microscope. Pieces of intestine pinned on balsa were incubated in oxygenated Krebs solution at 37 degrees C for 90-120 min and then fixed for 1 hr at room temperature in 4% formaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.4. The tissue was washed vigorously in several changes of 50% ethanol until the picric acid had been removed, stored overnight in phosphate buffer, and then exposed to 0.1% sodium cyanoborohydride in buffer for 30 min. Vasoactive intestinal peptide (VIP) was localized in separated layers containing myenteric plexus and longitudinal muscle using the peroxidase-antiperoxidase technique with imidazole intensification of the diaminobenzidine reaction product. At the light microscope level, tissue stained by this technique showed VIP-immunoreactive nerve cell bodies and processes throughout the thickness of the myenteric ganglia in numbers approximately equivalent to those seen in whole mounts processed by an established technique for the light microscopic demonstration of VIP, which does not involve exposure of tissue to glutaraldehyde. VIP-immunoreactive structures that were first identified at the light microscope level were subsequently examined at the electron microscope level. VIP-immunoreactive axon profiles were found to form synapses on both immunoreactive and nonimmunoreactive myenteric neurons. The fine structural appearance of the different cell types present in whole mount preparations prepared by this method was similar to that seen in conventionally fixed tissue, except that free and bound ribosomes were absent from the tissue processed for immunocytochemistry. The method described here is reliable and no more difficult than presently available methods for preembedding electron microscopic immunocytochemistry on sections. Its main advantage is that immunoreactive structures for ultrastructural study can be selected from the entire population of chemically identified nerves within a whole mount rather than from a smaller sample present within a section. This technique is applicable to other tissues that can be stained immunohistochemically in whole mounts. The fixation and penetration enhancement procedures can also be adapted for immunocytochemical studies on vibratome or frozen sections.     

Stabilization of cholesterol in myelin with digitonin: observation with polarized light.

Cholesterol can be preserved in tissues dehyrated for light microscopy by incorporating digitonin into glutaraldehyde fixation. Fresh and fixed frozen sections of rat sciatic nerve exhibit strong, radially positive birefringence when viewed with polarized optics. However, tissue fixed and dehydrated with standard methods for Epon embedding shows very weak radially positive birefringence. The use of digitonin with a variety of techniques generally permits retention of optical activity in embedded nerves. Such observations suggest a preservation of lipids (presumably cholesterol) in molecular orientations necessary to produce Maltese cross patterns.     

A Study of Criteria Permitting the Use of Plastinated Specimens for Light and Electron Microscopy

Plastination permits the preservation of anatomical specimens in a physical state approaching that of the living condition. We studied the possibility of using silicone plastinated fragments of spleen and pancreas for optical and electron microscopy, and found that with an adequate fixation protocol, plastinated specimens can be used for both light microscopy and ultra-structural studies. Deplastination with sodium methoxide permitted production of clean sections. Artifacts produced by plastination/deplastination could be nearly eliminated by glutaraldehyde/formaldehyde fixation. The (Biodur) silicone S10 polymer is transparent and stable in an electron beam, and plastinated tissues can be contrasted or colored similar to tissues embedded in Epon 812. In addition to being very life-like, plastinated tissues are stable and easy to handle. They can also be used for electron and light microscopic studies. This technique may also allow retrospective epidemiological studies of archived pathology specimens.     

Application of immunoelectron microscopy for identification of T lymphocyte subsets and HLA DR antigen positive cells in tissue

The identification of T lymphocyte subsets by means of monoclonal antisera in tissue has so far been restricted to light microscopic observations on cryostat sections, since the conventional fixatives as formaldehyde, B5, or glutaraldehyde seem to denature the surface antigens. Applying a mild fixative, periodate-lysine-paraformaldehyde (PLP), we were able to demonstrate T cell subsets on the electron microscopic level in human tonsils with minimal loss of antigenicity of the surface markers Leu 1, OKT4, OKT8, and HLA DR. The preservation of the tissue could be compared favorably to fixation with glutaraldehyde, and provided an ultrastructural basis to distinguish helper and suppressor cells by morphological features as well.     

Application of silver stains to gastric endocrine cells in plastic sections

Summary A simultaneous light and electron microscopic study of mouse gastric mucosa was made to determine whether the silver nitrate methenamine stain of Duk-Ho Lee could be used to stain gastric endocrine-like cells in plastic embedded tissue. Examination of consecutive thick and thin sections showed that this stain blackened the granules of the predominant type of endocrine-like cell present. Blackening of the granules with silver occured in tissue fixed in osmium tetroxide solution with or without dichromate salt or in tissue fixed in glutaraldehyde then treated with osmium. The intensity of staining was deepest in the osmium-dichromate fixed tissue, but the glutaraldehyde-osmium procedure gave less interference from diffuse silver impregnation and better preservation of detail for electron microscopy.     

Preservation of fine structure in vibratome-cut sections of the central nervous system stained for light microscopy

A technique without negative effects on tissue preservation that allows precise identification and subsequent removal of central nervous system nuclei for ultrastructural analysis is described. The procedure uses 200 microns thick Vibratome-cut sections of glutaraldehyde fixed brains. These sections are stained for 25 seconds with a methylene blue solution and stored for 4 hours in 0.2 M pH 7.4 phosphate buffer in 4% sucrose for optimal visualization at the light microscopic level. The stock solution of 1 g methylene blue and 1 g sodium borate in 100 ml of distilled water, is filtered through a Millipore filter and diluted 5:95 with distilled water immediately prior to use. Regions of specific interest are then processed for electron microscopy.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds.

Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study; I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32-34 s, final temperature between 40 degrees C and 47 degrees C.     

Light microscopic localization of cytochemical reactions in epoxy-embedded material for electron microscopy.

Tissues from mice were fixed in 1.5% glutaraldehyde, treated for the ultrastructural localization of alkaline phosphatase or Mg++-dependent adenosine triphosphatase, post-fixed in osmium tetroxide, dehydrated and embedded in plastic for electron microscopy. The sites of reaction were visualized in 1-mu plastic sections counterstained with toluidine blue, using a phase contrast microscope. The data show a close correlation between the sites of reaction observed with the phase contrast microscope and the sites studied with the electron microscope. The use of this technique for the study of these phosphatases in normal and pathologic tissues is recommended in order to achieve a high degree of accuracy in selecting a portion of the tissue sample for electron microscopy and to obtain greater resolution in the localization of these enzymes with the light microscope.     

Ultrastructural localization of nucleic acids through several cytochemical techniques on osmium-fixed tissues: comparative evaluation of the different labelings

Several cytochemical techniques, such as sodium tungstate, acid hydrolysis phosphotungstic acid (HAPTA), ethylenediaminetetraacetic acid (EDTA), RNase-gold, and osmium-ammine, have been applied for the ultrastructural demonstration of nucleic acids on sections of tissues fixed in glutaraldehyde postfixed with osmium tetroxide and embedded in Epon. In order to obtain specific results, the sections had to be treated with sodium metaperiodate prior to performing the labeling protocol. The results for each method were identical to those obtained on nonosmicated tissues; the main difference being the enhancement in the ultrastructural preservation, which allowed for higher resolution. In addition to these techniques, and for comparative evaluations, DNA was also revealed by the DNase-gold approach on nonosmicated tissue sections. The consistency in the results, obtained over the nucleus with either EDTA or the RNase-gold complex for revealing RNA and those obtained with either osmium-ammine or DNase-gold for revealing DNA, supports the high specificity of the RNase-gold, DNase-gold, and osmium-ammine techniques. Furthermore, these results demonstrate the possibility of performing various cytochemical techniques on tissues processed for routine electron microscopy.     

Cryostat sections for coexistence studies and preembedding electron microscopic immunocytochemistry of central and peripheral nervous system tissue

Perfusion-fixed tissue blocks were incubated in high molar sucrose solutions, shock frozen in melting isopentane, and sectioned on a conventional cryostat. Semithin sections (2-4 microns) alternatingly stained for parvalbumin and glutamate decarboxylase enabled us to demonstrate the coexistence of both antigens in the same cell. Thick sections (40 microns) of central and peripheral nervous system tissue were immunostained and processed for correlated light and electron microscopic studies. At the electron microscopic level, the preservation of ultrastructural features such as membranes and synaptic contacts was comparable to that normally seen in vibratome sectioned material. Hence, this technique can successfully be used for preembedding coexistence studies and electron microscopic preembedding immunocytochemistry when vibratome sectioning is problematic.     

Fixation of mucus on rainbow trout (Oncorhynchus mykiss Walbaum) gills for light and electron microscopy

Fixation of mucus for the assessment of biofilms and surface associated pathogens often involves complex and expensive techniques. Rainbow trout killed by an overdose of MS 222 had their gills removed and immersion-fixed gently in buffered glutaraldehyde containing 2% Alcian blue. Control tissues consisted of gills fixed in Alcian blue-free fixative. Trout were also killed and directly immersed in liquid nitrogen and the gills freeze-dried then vapour fixed with osmium tetroxide at −50° C. Following fixation gill tissue was processed for light and electron microscopy. A continuous and intact mucous coat was not detected on tissue fixed by conventional methods but the addition of Alcian blue to the fixative preserved an extensive mucous coat trapped between the lamellae and overlying the epithelia. Electron microscopic examination revealed that mucus preservation with the conventional fixative was poor and intermittent whereas the addition of Alcian blue to the fixative greatly enhanced the preservation of the branchial mucous coat. Mucus appeared as interdispersed flocculant material between the epithelial microridges and formed extensive superficial sheets over the epithelium. Freeze-dried/vapour-fixed gill tissue also provides excellent preservation of the integrity of branchial mucous coats, the mucus appearing as a continuous sheet over the filament and secondary lamellae. However, freeze-dried tissue fails to preserve sufficient cellular integrity for this technique to be useful for light or transmission electron microscopy. The potential for use of glutaraldehyde-Alcian blue fixed-gill tissue diagnostically and in research are discussed.     

An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.

Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described. To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixature is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

An improved timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues

Summary Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

Optimization of a histopathological biomarker for sphingomyelin accumulation in acid sphingomyelinase deficiency

Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy.     

Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system

A pre-embedding immunocytochemical technique is described for combined light and electron microscope study of peptidergic neurons in the central nervous system. The protocol is especially designed to overcome the sampling problems inherent in electron microscope study of structures, such as luteinizing hormone-releasing hormone (LHRH) neurons, that are scattered individually across large brain regions. The fixation methods outlined for several mammalian species include immersion and vascular perfusion with acrolein. Fine-structural preservation and LHRH immunoreactivity obtained with this fixative are compared to results with more conventional fixatives. Vibratome sectioning and a "pretreatment" regime, which prepare the tissues for immunocytochemistry, are described. Immunocytochemical labeling is done with free-floating sections and the peroxidase-antiperoxidase unlabeled antibody enzyme technique. Techniques are also described for the subsequent processing of immunoreacted sections for electron microscopy. These methods ensure that the processed sections are readily scanned by light microscopy, so that regions containing immunoreactive structures can be specifically chosen for electron microscope analysis. Sample electron micrographs are shown that illustrate some fine structural features of LHRH neurons in rats, bats, ferrets, and monkeys, as revealed with the techniques described.     

High-resolution light microscopy (HRLM) and digital analysis of Pompe disease pathology.

Pompe disease is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase, responsible for the degradation of lysosomal glycogen. Absent or low levels of the enzyme leads to lysosomal glycogen accumulation in cardiac and skeletal muscle cells, resulting in progressive muscle weakness and death from cardiac or respiratory failure. Recombinant enzyme replacement and gene therapy are now being investigated as treatment modalities for this disease. A knockout mouse model for Pompe disease, induced by the disruption of exon 6 within the acid alpha-glucosidase gene, mimics the human disease and has been used to evaluate the efficacy of treatment modalities for clearing glycogen. However, for accurate histopathological assessment of glycogen clearance, maximal preservation of in situ lysosomal glycogen is essential. To improve retention of glycogen in Pompe tissues, several fixation and embedding regimens were evaluated. The best glycogen preservation was obtained when tissues fixed with 3% glutaraldehyde and postfixed with 1% osmium tetroxide were processed into epon-araldite. Preservation was confirmed by staining with the Periodic acid-Schiff's reaction and by electron microscopy. This methodology resulted in high-resolution light microscopy (HRLM) sections suitable for digital quantification of glycogen content in heart and skeletal muscle. Combining this method of tissue fixation with computer-assisted histomorphometry has provided us with what we believe is the most objective and reproducible means of evaluating histological glycogen load in Pompe disease.     

Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue.

Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5--01%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.     

The use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding.

Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or tricholoracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green-pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.