Immunohistochemical myofiber typing and high-resolution myofibrillar lesion detection in LR white embedded muscle

We have developed a method of fixing, embedding, sectioning, and staining that allows high-resolution detection of myofibrillar structure and myosin immunocytochemical muscle fiber typing in serial semithin sections of LR White plastic embedded muscle at the light microscopic level. Traditional approaches, such as cryostat sections, permit fiber typing, but small myofibrillar lesions (1-3 sarcomeres) are difficult to detect because of section thickness. Semithin sections of hydrophobic resins do not stain well either histochemically or immunocytochemically. Electron microscopy can resolve lesions and discriminate fiber types based on morphology, but the sampling area is small. Our goal was to develop a rapid method for defining both fiber type and high-resolution primary myofibrillar lesion damage. Mild fixation (1-4% paraformaldehyde, 0. 05-0.1% glutaraldehyde) and embedment in a hydrophilic resin (LR White) were used. Myofibrillar structure was extremely well preserved at the light microscopic (LM) level, and lesions could be readily resolved in Toluidine blue stained 500-nm sections. Fiber type was defined by LM immunomyosin staining of serial plastic semithin sections, which demonstrated reciprocal staining patterns for "fast (Sigma M4276) and "total" (skeletal muscle) myosins (Sigma M7523).     

Staining sections of water-miscible resins 2. Effects of staining-reagent lipophilicity on the staining of glycol-methacrylate-embedded tissues

Glycol methacrylate (GMA) sections of animal tissues were stained with a group of twenty-seven reagents of very varied chemical characteristics. The artefactual background staining of the resin was found to be dependent on the hydrophilic/lipophilic character of the staining reagent, as estimated from the logarithm of its octanol-water partition coefficient (log P). Intense background staining occurred with lipophilic stains, whose log P>2. In keeping with this, use of GMA semi-permeable membranes for enzyme histochemistry failed to give staining when using a lipophilic substrate, probably because the substrate was trapped in the membrane. An analysis of other routine histochemical stains—in terms of the probable occurrence of high resin background staining and low tissue sensitivity—is made. A numerical guide is provided to help avoid artefacts resulting from hydrophobic and size effects. Note: small, hydrophilic reagents (log P <0; molecular weight < 550 Da) are least likely to show either type of artefact. Conversely, reagents which are lipophilic, or/and of intermediate size (log P > 2; 550 < ionic weight < 1000 Da), give strong background staining.     

Oleic acid‐added embedding medium for histological analysis of hard tissue

For the histological analysis of hard tissue such as bone, various acrylate‐based materials have been used as an embedding medium. However, commercial embedding media are expensive, and cutting the embedded block takes a long time. In this study, mixtures of methyl methacrylate (MMA), di‐butyl‐phthalate (DBP), and oleic acid (OA) were tested for possible application as an embedding medium for large and small undecalcified bone specimens. Mechanical properties were tested in a compressive mode. We investigated the change of hydrophilicity in the sectioned surface by measuring the contact angle depending on the OA. Crystallinity was analyzed using a X‐ray diffractometer (XRD). Surface analysis was performed using a confocal laser scanning microscope. To determine the staining efficiency of staining dyes, hamatoxylin‐eosin (H&E) and Masson's trichrome (MT) staining methods were performed for the histological analysis of bone‐implant complex. We confirmed that the investigated embedding media showed good properties such as optimal mechanical strength appropriate for cutting the embedded block and proper staining efficiency for histological analysis. Therefore, the MMA/DBP/OA mixtures can be used as an embedding media appropriate for various hard tissues and bone‐implant complex. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

Stevenel's Blue, an excellent stain for optical microscopical study of plastic embedded tissues

Stevenel's Blue is a reliable, rapid, and clean, one-step polychromatic stain for 1 micron thick epoxy sections. The staining solution, originally used by L. Stevenel (1918) to stain human parasites, is made by adding diluted potassium permanganate (2%) to an aqueous solution of the methylene blue (1.3%) and redissolving the precipitate thoroughly, by boiling in water bath and filtering. Staining is carried out in a Coplin jar at 60 degrees C for approximately 10 minutes for tissues embedded in Epon 812 or Poly 812, or 20 minutes for tissues embedded in Spurr's medium. The sections are rinsed, air dried, and mouted in Permount. The staining solution is very stable, and does not tend to form precipitates on the tissue. The stain brings excellent histological differentiation to nuclear, cytoplasmic, and extracellular components. Incorporation of the stain by elements within each tissue varies from intense to light with a subtle gradation of intermediate shades of purple and blue tones. For most cell structures the density of the stain parallels the electron density of that structure as seen under the electron microscope. For example, nucleoli and heterochromatin stain in dark purple while euterochromatin appear in a light blue shade. In all cases, the embedding media remains unstained. The bond between Stevenel's Blue and the tissues is stable, remaining unaltered by the mounting medium. It is also resistant to time-fading.     

A double lead stain method for enhancing contrast of ultrathin sections in electron microscopy: a modified multiple staining technique

A modification of the conventional method for the staining of ultrathin sections resulted in an increase in contrast of ultrastructural detail in tissues. Tissues embedded in Spurr's low viscosity embedding medium were stained with freshly centrifuged Reynolds' lead citrate for 1–5 min, rinsed in double distilled water and dried prior to staining with a saturated solution of uranyl acetate for 40 min, and freshly centrifuged Reynolds' lead citrate for 20 min. Sections treated by this procedure showed enhanced staining of cellular organelles and cytoplasmic matrix. This procedure is recommended for tissues with poor staining qualities resulting from either prolonged fixation or from inadequacies in the buffer or embedding medium used.     

Labelled-replica techniques: post-shadow labelling of intramembrane particles in freeze-fracture replicas

Three methods are described for direct post-fracture, post-shadow labelling of individual classes of intramembrane particles (IMPs) in freeze-fracture replicas of biological membranes. The P-face IMPs corresponding to the acetylcholine receptor complexes (AChRs) of vertebrate neuroeffector junctions are identified by post-replication labelling with ferritin-antibody complexes and with neurotoxin-biotin-avidin-colloidal gold affinity ligands. (The freeze-etch nomenclature of Branton et al., 1975, is used in this report.) These post-shadow labelling techniques resemble conventional en bloc labelling techniques except that the labelling reagents must penetrate a thin but discontinuous layer of platinum superimposed on the molecules of interest. In the ‘sectioned labelled-replica technique’, the replicated and labelled tissues are stained, embedded in plastic and sectioned parallel to the replica-tissue interfaces. In the direct ‘labelled-replica techniques’, the replicated and labelled samples are freeze-dried or critical point dried, the labelled surfaces are stabilized by carbon coating, and the underlying tissues are dissolved, allowing the labelled-replicas to be examined as conventional freeze-fracture replicas. The unshadowed side of each AChR IMP is shown to retain sufficient biochemical information to permit both immunospecific and neurotoxin specific labelling despite formaldehyde fixation, freezing, fracturing, platinum shadowing, and thawing in aqueous media. A new mixed ferricyanide-osmium staining method reveals electron opaque structures spanning the membrane bilayer in the same size, number and distribution as the labelled IMPs. These experiments demonstrate the feasibility of identifying individual IMPs in freeze-fracture replicas and may allow the identification of specific membrane lesions in human disease.     

A technique for obtaining ribbons of epoxy and other plastic sections for light microscope histology

A method is described by which ribbons of thick, large area sections of material embedded in epoxy resins prepared from standard recipes for electron microscopy, can be cut using conventional microtomes. The epoxy blocks are double embedded in an epoxy/polyethylene glycol mixture and ribbons are cut dry with fluorocarbon coated long-edged (‘Ralph’) glass knives. The method can also be applied to other plastic embedding media such as glycol methacrylate.     

Staining sections of water-miscible resins

Penetration of hydrophilic acid and basic dyes into sections cut from glycol methacrylate (GMA)-embedded tissues was studied; as were the effects on such staining of superficial coatings of thin layers of GMA. Dye size was a major factor in controlling penetration of resin and staining of tissues. ‘Large’ dyes (>1000 Da) entered GMA very slowly, and only stained those tissue components poorly infiltrated by resin. ‘Small’ dyes (< 550 Da) penetrated GMA readily, and stained tissue components whether or not they were resin-infiltrated. Dyes of intermediate size penetrated the resin, but the staining of resin-infiltrated tissue elements was slow. Background staining of resin also varied with dye size. Large dyes gave no staining of GMA. Small dyes did, but were readily removed by water washing. Dyes of intermediate size penetrated resin slowly, and once inside were lost slowly. This gave background staining which required use of the plasticizing solvent ethanol for its removal. Increases in resin cross-linking also reduced staining rates. As a consequence, it is possible to predict the probable suitability, or otherwise, of various staining reagents proposed for use with GMA sections; and also the probable influences of histoprocessing on stain penetration. In particular it is suggested that penetration of colloidal metals and macromolecular reagents (e.g. labelled antibodies and lectins) will be limited to resin-free structures, and to the surface of resin sections. The use of superficial GMA coatings as convenient semipermeable membranes for enzyme histochemistry is also noted.     

Preparing sections of skeletal muscle for transmission electron analytical microscopy (TEAM) of diffusible elements.

Comparative morphological examination and elemental analysis were carried out in structural compartments of sections of skeletal muscles. These had been prepared either by conventional plastic embedding technique or by various methods of cryo-ultramicrotomy. The analyses were performed in a Philips EM 301 with an Edax energy-dispersive X-ray spectrometer. Spectra obtained from sections of plastic-embedded muscle depended on the reagents used for fixation and staining and were absent if these were omitted. Brief fixation with glutaraldehyde resulted in gross ionic changes, and sectioning of frozen material with trough liquid led to extraction of elements. Sections cut from unfixed and frozen muscle without trough liquid showed numerous peaks. (Mg, P, S, Cl, K, Ca). In the superficial parts of the fibres of freeze-dried sections reproducible spectral differences were found between different structures. Thus, rapid freezing of unfixed tissue, dry cutting in the frozen state, and freeze-drying should be the procedure of choice if data on diffusible ions are desired.     

Estimating the effect of etching agents on plastic sections

The effects on epoxy embedding media of certain etching procedures have been studied in two ways. Firstly the resin present in thick sections, before and after etching, was demonstrated by staining the plastic with Sudan Black B. Secondly the change in weight of plastic blocks after their exposure to etching procedures was measured. Methods involving halogens in acetone, alkoxides in ethanol or in methanol-benzene, and performic acid resulted in gross removal of plastic. Additionally, in keeping with the widespread belief that etching may increase permeability of plastic sections to stains, most of the etch methods studied, including organic solvents alone, did remove some plastic from the blocks.     

Ponceau 2R staining on semi-thin sections of tissues fixed in glutaraldehyde-osmium tetroxide and embedded in epoxy resins

Proteins were stained (brilliant red) by using 0·5% Ponceau 2R (C.I. 16150) in sulphuric acid (pH 2) at 40°C on sections 1–2 μm thick of tissues fixed in glutaraldehyde-osmium tetroxide and embedded in epoxy resins. The staining is free from precipitates and does not require the removal of the embedding media. Section bleaching with 1–2% periodic acid at 40°C for 30–90 min was also carried out in order to obtain a more brilliant colour of the proteins which may be photographed using black and white films.     

The influence of the embedding medium when staining for electron microscopy: the penetration of stains into plastic sections.

Using a sectioned-section procedure it was found that only a few structures, e.g. the connective tissue elements, were deeply penetrated by phosphotungstic acid (PTA) and uranyl acetate (UA). Reynold's lead citrate appeared to penetrate more generally. Using stains selective for the embedding medium, and also observing the surfaces of shadowed sections, it was concluded that structures readily penetrated by polar stains were only poorly infiltrated by non-polar embedding media. It is argued that this differential infiltration leading to differential penetration by polar stains largely controls the pattern of staining of ultrathin sections by PTA and UA.     

A simple method for exposing plastic embedded structures for scanning electron microscopy

Surfaces of structures which may be identified in sections of plastic embedded tissue can be exposed for examination using scanning electron microscopy by removal of the embedding medium from the sectioned block with sodium methoxide. Correlation of the information provided by sections and scanning electron microscopy is therefore possible. This technique has been used in examining the developing peristome teeth of the moss Funaria hygrometrica.     

Postembedment staining of complex carbohydrates: Influence of fixation and embedding procedures

In recent years technological advancements have led to improvements in ultrastructural cytochemical methods for localizing and characterizing complex carbohydrates. In particular the introduction of lectins with specific affinities for various sugars and sugar sequences as histochemical probes has increased knowledge concerning the cellular and subcellular distribution of glycoconjugates. Development of nonepoxy-based embedding materials has provided increased sensitivity compared to the earlier less specific methods and the current lectin methods for localizing sugar moieties. Postembedment staining based on the reactivity of functional groups present in sugars, such as hydroxyl groups, vicinal diol groups, carboxyl groups, and sulfate esters, requires specific conditions for tissue fixation and embedding. The same requirements pertain to staining based on lectin binding. The influence of fixation and embedment using older and newly developed embedding mixtures on the ultrastructural demonstration of complex carbohydrates is considered in this discussion. Fixation with osmium tetroxide and embedment in epoxy resins provides the least sensitive combination for the detection of the reactive groups of complex carbohydrates. The best ultrastructural demonstration of glycoconjugates is achieved when nonosmicated tissues are embedded in nonepoxy resins.     

Cryoultramicrotomy versus plastic embedding: comparative immunocytochemistry of rat anterior pituitary cells

The anterior pituitary of the rat is used as a model for the study of the effects of freezing or plastic embedding on the maintenance of antigenicity. Rat anterior pituitaries are fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer pH 7.4. Some of the blocks are post-fixed before being divided into two lots. One batch is frozen, while the other is dehydrated and embedded. The indirect antibody enzyme method is applied to ultrathin sections obtained by cryoultramicrotomy after freezing or by sectioning after embedding. All six pituitary hormones are detected by both methods. Comparison shows that the morphological characteristics are identical for both techniques, though ultrastructural preservation is better after embedding. Immunoreactivity is found in secretory granules and sometimes in the endoplasmic reticulum. Osmium postfixation may reduce or even abolish antigenicity in plastic-embedded tissue. After cryoultramicrotomy, however, even after osmium fixation, antibody may be used 1000 times more diluted than after plastic embedding. Embedding preserves ultrastructure and limited antigenicity while the use of cryoultramicrotomy is a far more sensitive technique.     

Modified paraffin wax for improvement of histological analysis efficiency

Paraffin wax is usually used as an embedding medium for histological analysis of natural tissue. However, it is not easy to obtain enough numbers of satisfactory sectioned slices because of the difference in mechanical properties between the paraffin and embedded tissue. We describe a modified paraffin wax that can improve the histological analysis efficiency of natural tissue, composed of paraffin and ethylene vinyl acetate (EVA) resin (0, 3, 5, and 10 wt %). Softening temperature of the paraffin/EVA media was similar to that of paraffin (50–60°C). The paraffin/EVA media dissolved completely in xylene after 30 min at 50°C. Physical properties such as the amount of load under the same compressive displacement, elastic recovery, and crystal intensity increased with increased EVA content. EVA medium (5 wt %) was regarded as an optimal composition, based on the sectioning efficiency measured by the numbers of unimpaired sectioned slices, amount of load under the same compressive displacement, and elastic recovery test. Based on the staining test of sectioned slices embedded in a 5 wt % EVA medium by hematoxylin and eosin (H&E), Masson trichrome (MT), and other staining tests, it was concluded that the modified paraffin wax can improve the histological analysis efficiency with various natural tissues. Microsc. Res. Tech. 73:761–765, 2010. © 2010 Wiley‐Liss, Inc.     

Triton X-100 pretreatment of LR-white thin sections improves immunofluorescence specificity and intensity

The staining of intracellular antigenic sites in postembedded samples is a challenging problem. Deterioration of antigenicity and limited antibody accessibility to the antigen are commonly encountered on account of processing steps. In this study preservation of the antigen was achieved by fixing the tissues with mild fixatives, performing partial dehydration, and embedding in a low crosslinked hydrophilic acrylic resin, LR-White. Permeabilization of cell membranes with Triton X-100 is well documented but can affect some antigen conformations. We tested the effect of Triton X-100 on the ED1 antigen present in the lysosomal membrane of the macrophage in cell culture. The ED1 antigen in the lysosome was resistant to extraction by Triton X-100. Interestingly pretreating the LR-White sections of macrophage pellets with Triton X-100 improved the staining intensity of ED1. The most intense and clear specific fluorescent staining was observed when sections were pretreated with 0.2% Triton X-100 for 2 min. Longer exposure of sections to 0.2% Triton or 2 min exposure to 2% Triton lead to reduced ED1 labeling. SEM observations indicated that the detergent extracted a component from the cells and not the resin and was determined to be lipid. This novel technique could be applied in many research areas where postembedding fluorescent immunolabeling with higher labeling intensity is desired.     

Why low temperature embedding for X-ray microanalytical investigations?

Freeze-drying followed by infiltration with resin and polymerization by UV light at low temperatures and under constant vacuum conditions is an alternative tissue preparation technique for microprobe analysis. Embedding is carried out with the nonpolar low-temperature embedding resin (Lowicryl HM20) which allows infiltration and polymerization at temperatures down to ?50°C. Sections of low temperature embedded material can be cut dry at ?60°C or at room temperature. Sectioning at low temperatures is an alternative for preparations that are difficult to cut at room temperature. The morphological preservation is adequate for the identification of structures such as mitochondria, lysosomes and different types of endoplasmic reticulum in liver cells. Some physical properties of Lowicryl resins, such as mass loss under the electron beam and high contrast, are positive characteristics for the analysis of semi-thick sections. No significant differences in the elemental composition could be detected between tissue which was freeze-dried or freeze-substituted prior to embedding. Freeze-drying is less time consuming. By avoiding contact with organic solvents the risks of ion loss and redistribution are diminished. In contrast to freeze-dried thin cryosections, low temperature embedded material can be sectioned for light microscopy and areas of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogeneous cell populations. The initial preparative step—the cryofixation— determines to a high degree the morphological preservation of freeze-dried and embedded tissue.     

Methods for electron microscopy of the lamellated osmiophilic bodies of the lung

A study has been made of methods of fixing, staining, and embedding for electron microscopy the lamellated osmiophilic bodies (LOPBs) of the type II cells of the lung, which are probably connected with the lung surfactant. The preferred method for fixing and staining uses successively 2% glutaraldehyde, 1% osmium tetroxide, and a mixture of 10 vol M/40 lead nitrate with 10.5 vol M/60 potassium ferricyanide: all reagents are in cacodylate buffer. Unless the ferri-cyanide is in excess, the sections show blotchy precipitation. The bodies are damaged by ethanol and 1:2 epoxypropane. Dioxan or (with a quick time schedule) acetone may be used as a dehydrant and thinner prior to Araldite embedding. Durcupan water-soluble resin may also be used for dehydration and embedding; the preferred embedding mixture is: 5 ml resin A, 11.7 ml hardener B, 1.5 ml hardener C, and 0.4 ml plasticizer D, cured for 24–48 h at 60°C. Examination of LOPBs on a goniometer stage shows that they are composed of layers spaced at 4 nm, each lamella containing several such layers. Further evidence is adduced linking the LOPBs with the surfactant, and the application of the new methods to various problems involving the LOPBs is discussed.     

Difference in hydrophobic and hydrophilic multilayered systems

Functional clothing, sportswear and military uniforms, are mainly made of polyester or cotton fabrics for dealing with sweat absorption and heat transfer produced by the human body. The human body typically wears two or three garments, such as innerwear, T-shirt, and jacket, thus comprising a multilayered system. Garments’ responses to heat transfer and vapor flow differ depending on whether their physical structures are hydrophobic or hydrophilic. Hence, this study analyzes differences in heat transfer and vapor flow, induced by the human body covered with multilayered garment systems, consisting of three polyester or cotton layers. In particular, it verifies three differences in the heat transfer and relative humidity, amount of relative humidity, and response time of thermal equilibrium in multilayered systems by using a new measurement system, “H&M evaluation device”. Resultant data provide precise information regarding several differences in hydrophilic and hydrophobic multilayered fabric systems.