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.     

Staining of DNA-phosphate groups and DNA-aldehyde molecules with dyes of the azine group

The investigation reports on the use of safranine-SO2 and phenosafranine-SO2, prepared with N HCl or oxalic acid plus potassium metabisulphite, for staining rat liver sections following Feulgen procedure. It has been found that optimum staining of DNA-aldehyde molecules is possible with safranine-SO2 and phenosafranine-SO2, prepared with N HCl and potassium metabisulphite, upto a duration of one week after the preparation of the dye-reagents. Thereafter, staining intensity of the nuclei produced by the dye-reagents is gradually diminished. Staining of acid-hydrolysed sections is also possible with aqueous solutions of these dyes. Moreover, DNA-phosphate groups can also be stained with aqueous solutions of these dyes after selective extraction of RNA with cold phosphoric acid. The in situ absorption spectra of nuclei, stained for DNA-aldehyde molecules with safranine-SO2, phenosafranine-SO2 and aqueous solutions of these dyes, have been presented in this paper. Also presented herein are absorption data of nuclei stained with these dyes after selective extraction of RNA. It has been found that absorption-peaks of nuclei stained differently are different from one another. The implications of these findings have been discussed.     

Hoffman's violet and dahlia as specific stains for animal chromosomes.

The paper deals with staining of the chromosomes of animal testicular materials with two basic dyes, Hoffman's violet and dahlia of the triphenylmethane group, following iodine-dye procedure. The important finding, as presented herein, is that iodinated alcohol after staining can be substituted with various acids, both organic as well as inorganic, all of which act as trapping agent preventing leaching of the dye that binds with the chromosomal DNA. It is clear from this study that RNA is not involved by this process of staining, since treatment of stained sections with cold phosphoric acid at 5 degrees C for 20--25 min and then stained also reveals perfect colouration of the chromosomes without any cytoplasmic staining. The in vitro absorption properties of Hoffman's violet have also been presented herein. The probable mechanism of action of these dyes has been suggested.     

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.     

Microwave-assisted rapid plant sample preparation for transmission electron microscopy

The preparation of plant leaf material for transmission electron microscopical investigations can be a very time- and labour-consuming task as the reagents infiltrate the samples quite slowly and as usually most steps have to be performed manually. Fixation, buffer washes, dehydration, resin infiltration and polymerization of the resin-infiltrated leaf samples can take several days before the specimen can be cut ultrathin and used for ultrastructural investigations. In this study, we present a microwave-assisted automated sample preparation procedure that reduces preparation time from at least 3 days to about 5 h – with only a few steps that have to be performed manually – until the plant sample can be ultrathin sectioned and observed with the transmission electron microscope. For studying the efficiency of this method we have compared the ultrastructure of different leaf material ( Arabidopsis thaliana , Nicotiana tabacum and Picea abies ) which was prepared with a conventional, well-established chemical fixation and embedding protocol and a commercially available automated microwave tissue processor. Despite the massive reduction in sample preparation time no negative effects on cutting properties of the blocks, stability of the sections in the electron beam, contrast and ultrastructure of the cells were observed under the transmission electron microscope when samples were prepared with the microwave-assisted protocol. Additionally, no negative effects were detected on the dimensions of fine structures of grana stacks (including membranes, inter- and intrathylakoidal spaces), the nuclear envelope and the plasma membrane as the diameter of these structural components did not differ between leaf samples (of the same species) that were processed with the automated microwave tissue processor or by conventional fixation and embedding at room temperature.     

Two rapid and simple methods used for the removal of resins from 1.0 micron thick epoxy sections.

Ultrathin epoxy sections are commonly used in electron microscopy. More useful information can often be obtained by examining thicker 0.5--3.0 micron) resin sections under the light microscope. However, there are some histological stains that are unable to penetrate these resins. This short note describes the easy and rapid preparation of two resin-removal solutions which allow subsequent staining of 1.0 micron thick sections without obvious tissue damage and with excellent results using standard histological dyes.     

A simple method of preparing 1–2 μm sections of large tissue blocks using glycol methacrylate

The method described allows the production of 1–2 μm sections of large tissue blocks. Sections can be obtained from conventional routine histological microtomes. The hydroxyethyl methacrylate is compatible with a wide range of fixatives and a variety of standard histological staining techniques may be applied. Small biopsies, e.g. renal, liver, jejunal etc., can be processed cut and stained within 24 h of receipt. Blocks and stained sections show no apparent deterioration over the 4 years in which the technique has been employed in this laboratory.     

LR White sections as slot grid support films for transmission electron microscopy

The utility of LR White sections as slot grid support films for the examination of thin resin‐embedded tissue sections by transmission electron microscopy was investigated and compared with traditional formvar‐carbon films. Throughout a variety of staining procedures, which involved the use of organic solvent, oxidizing agents, strong acid and prolonged incubation, LR White support films remained intact and the attached tissue sections remained adherent. By contrast, complete loss of formvar‐carbon support films occurred in 25% of preparations during routine staining with aqueous reagents. This loss increased to 62% following staining with either alcoholic or oxidizing and acidic stains, and to 66% following prolonged (immunohistochemical) staining. Tissue contrast, ultrastructural detail and immunohistochemical staining intensity were comparable between sections on the two types of support film. The use of LR White sections as support films for slot grids represents a quick, cheap, simple and robust alternative to traditional support films and, furthermore, requires no carbon coating     

Evaluation of fluorescent dyes for epoxy impregnation of porous ceramics

Eight different commercially available fluorescent dyes (fluorochromes) were tested for suitability for use in low-viscosity epoxy resin. Dyes were compared based on solubility in different solvents and epoxy resin and a numerical criterion for each dye's fluorochromicity. The two best dyes, based upon the brightness of each dye after illumination by a UV source, were Hostasol Red GG and Hostasol Yellow 3G. These two dyes in epoxy resin were used to visualize impregnation and remnant porosity in porous superconductor ceramic pellets. The impregnant was either cured epoxy or a low melting point alloy.     

Preliminary studies of the effects of DMSO on histochemical reactions

Slices of mammalian kidney were treated with concentrations of dimethyl sulphoxide (DMSO) theoretically high enough to protect living cells against damage during freezing and thawing. The blocks were cooled slowly to ?45°C and then quenched in liquid nitrogen. Sections were cut at ?45°C and post-fixed in various reagents. They were stained for lysosomal and mitochondrial enzymes. Cellular and tissue morphology was better preserved than in tissue which had been treated by conventional histochemical freezing methods. The staining of the enzymes was satisfactory and the localization of mitochondrial enzymes, in particular, was sharper.     

Studies in fluorescence histochemistry: II. The demonstration of periodate-reactive mucosubstances with pseudo-Schiff reagents*

According to Kasten, periodate-oxidized mucosubstances which have been stained with his fluorescent Schiff-type reagents (solutions of basic fluorescent dyes saturated with sulphur-dioxide) emit a specific fluorescence in sections of fixed tissue. In my experience it is sometimes difficult to observe this fluorescence because it is obscured by the similarly-coloured fluorescence emitted by proteins and nuclei. This and other difficulties, both theoretical and practical, can be mostly overcome by methylating tissue sections beforehand with a methanolic solution of thionyl chloride, then oxidizing them in periodic acid, and finally treating them first with sulphurous acid (at pH 3) and second with a solution of any basic fluorescent dye at a pH between 2 and 3. In sections treated in this way, nearly all periodate-oxidized mucosubstances emit a much more intense fluorescence than when treated with Kasten's Schiff-type reagents; and furthermore, that such mucosubstances fluoresce at all lends support to the theory that aldehydes react with Schiff's reagent proper not through N-sulphinic acids, as has been commonly supposed, but by reacting first with the sulphurous acid present in Schiff's reagent to form an intermediate alkyl sulphonic acid which then combines with basic pararosaniline (the essential ingredient of Schiff's reagent) to give the familiar magenta-coloured product.     

Use of aqueous solutions of two basic dyes for the demonstration of DNA

This paper presents informations as to the ability of aqueous solutions of two basic dyes, such as Dahlia and Victoria blue, belonging to aminotriarylmethane group for the staining of DNA-aldehyde molecules as well as DNA-phosphate groups. It has been found that sections of rat tissues stained with aqueous solutions of these dyes after acid hydrolysis followed by drying between folds of filter paper and treatment in n-butanol for a minute and then by a very brief treatment in a mixture consisting of equal parts of n-butanol and absolute ethanol reveal well-stained nuclei. Tissue sections after acid hydrolysis when stained with aqueous solutions of these dyes and then treated with SO2 water do not reveal any colouration of the nuclei. Since both the dyes are without any primary amino group in their molecules, it has been concluded that the imino group of Dahlia and the tertiary amino group of Victoria blue with cold concentrated phosphoric acid and then stained with any of these dyes also exhibit well-stained nuclei. The absorption spectra of nuclei stained with these dyes for DNA-aldehyde molecules as well as DNA-phosphate groups reveal positions of the peaks of maximum absorption at the same wavelength, which, however, are different in the case of nuclei stained with the two dyes. The implications of these findings have been discussed.     

Imaging thin and thick sections of biological tissue with the secondary electron detector in a field-emission scanning electron microscope

A field-emission scanning electron microscope (FESEM) equipped with the standard secondary electron (SE) detector was used to image thin (70–90 nm) and thick (1–3 μm) sections of biological materials that were chemically fixed, dehydrated, and embedded in resin. The preparation procedures, as well as subsequent staining of the sections, were identical to those commonly used to prepare thin sections of biological material for observation with the transmission electron microscope (TEM). The results suggested that the heavy metals, namely, osmium, uranium, and lead, that were used for postfixation and staining of the tissue provided an adequate SE signal that enabled imaging of the cells and organelles present in the sections. The FESEM was also used to image sections of tissues that were selectively stained using cytochemical and immunocytochemical techniques. Furthermore, thick sections could also be imaged in the SE mode. Stereo pairs of thick sections were easily recorded and provided images that approached those normally associated with high-voltage TEM.     

Oolong tea and LR‐White resin: a new method of plant sample preparation for transmission electron microscopy

Simplifying sample processing, shortening the sample preparation time, and adjusting procedures to suitable for new health and safety regulations, these issues are the current challenges which electron microscopic examinations need to face. In order to resolve these problems, new plant tissue sample processing protocols for transmission electron microscopy should be developed. In the present study, we chose the LR‐White resin‐assisted processing protocol for the ultrastructural observation of different types of plant tissues. Moreover, we explored Oolong tea extract (OTE) as a substitute for UA in staining ultrathin sections of plant samples. The results revealed that there was no significant difference between the OTE double staining method and the traditional double staining method. Furthermore, in some organelles, such as mitochondria in root cells of tomatoes and chloroplast in leaf cells of watermelons, the OTE double staining method achieved little better results than the traditional double staining method. Therefore, OTE demonstrated good potentials in replacing UA as a counterstain on ultrathin sections. In addition, sample preparation time was significantly shortened and simplified using LR‐White resin. This novel protocol reduced the time for preparing plant samples, and hazardous reagents in traditional method (acetone and UA) were also replaced by less toxic ones (ethanol and OTE).     

Specific staining of nuclei with aqueous solutions of celestin blue B and gallocyanine

This paper presents methods for specific staining of nuclei with aqueous solutions of celestin blue B and gallocyanine in tissue sections from which RNA has been extracted selectively with concentrated phosphoric acid at 5 degrees C for 20 min or by hydrolysis in 6 N HCl at 28 degrees C for 15 min. It has been found that pH of the freshly prepared celestin blue B dye solution is 3.0 and that of an aqueous solution of gallocyanine is 2.8. These pHs can be lowered to 1.5 with concentrated sulphuric or nitric acid and at this pH staining of the nuclei is possible. But with concentrated sulphuric or nitric acid and at this pH staining of the nuclei is possible. But if the pHs are lowered with concentrated hydrochloric or phosphoric acid, effective use of these dyes is not possible. It has been suggested that some dispersion of the two dyes takes place with concentrated sulphuric or nitric acid which are used to lower the pH. Staining of the nuclei is also possible with an aqueous solution of celestin blue B at pH 3.0 but the same is not possible with gallocyanine at pH 2.8. The absorption spectra of nuclei stained with an aqueous solution of celestin blue B at pH 1.5 and 3.0 are fairly identical, the peak of maximum absorption being at 620 nm. Those of nuclei stained with an aqueous solution of gallocyanine reveal irregular peaks. Possible implications of these findings have been discussed.     

An immunofluorescence method for postembedded tissue in the acrylic resin Technovit 9100 New® using fluorescein isothiocyanate secondary detection

Immunofluorescence labeling on postembedded tissue in resin is a formidable task. Although resin components and stabilizers are a source of additional strong native fluorescence that overlaps with absorption and emission spectra of commonly used green fluorophores, the unfixed tissue is also subject to native fluorescence. For tissue embedded in resin, we hypothesized that initially removing the resin and subsequently quenching the native fluorescence from the sample could result in specific immunofluorescence signals. The hypothesis was tested on fixed tissue samples embedded in Technovit 9100 New®. Deacrylated and rehydrated semithin sections from a variety of soft tissues were exposed to a quenching solution prior to immunolabeling. Cryostat sections from snap frozen tissue were also stained to assess whether all antigens investigated in fixed tissue were adequately detected. The secondary detection included antibodies conjugated with fluorescein isothiocyanate. The results were evaluated using conventional dark-field and confocal laser scanning microscopy. Both forms of microscopy confirmed the considerable lowering of the native fluorescence associated with the resin and fixed tissue samples with enhanced specific signal. The cryostat tissue sections using the same antibodies in equivalent concentrations confirmed labeling of the same cellular sites as those observed in the fixed tissue. This article describes a method for immunofluorescence labeling in Technovit 9100 New resin embedded tissue and suggests the likely chromogenic elements generating autofluorescence. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

An investigation of the fixation and staining of lipids by a combination of malachite green or other triphenylmethane dyes with glutaraldehyde

Mixtures of the monocationic triphenylmethane dyes, malachite green or crystal violet, with glutaraldehyde, retained and stained phospholipid droplets in chloroplasts of leaves of Lolium multiflorum Lam. These dyes also stained trilinolenin; phosphatidic acid dioleoyl, dipalmitoyl; phosphatidylcholine dioleoyl, dilinoleoyl; and phosphatidylethanolamine dioleoyl on filter paper models. In this model system the dipalmitoyl derivatives of phosphatidylcholine and phosphatidylethanolamine did not stain well, if at all. Washing of the dyed samples with 0.5 M sodium chloride solution did not remove the colour, suggesting that the interaction is unlikely to be purely ionic. Except with trilinolenin, the colour and possibly the lipid samples were removed from the filter paper model system on washing with 100% ethyl alcohol. Other triphenylmethane dyes (methyl green, light green and fast green FCF) did not retain phospholipid droplets in tissue. Fast green did, however, stain phospholipids in the model system. Two quinone-imine dyes, neutral red and toluidine blue O, while staining phospholipids in the model system did not retain droplets on the chloroplasts but did assist in the retention and staining of cell membranes. The basis of the reaction between lipid and dye is discussed in relation to the structural formulae of the dyes and model lipids. It is possible that there is an interaction between the hydrophobic fatty acid ester side chains of the lipid and the dyes. Neither the phosphate nor the polyhydric alcohol moieties of the lipid seem to be essential for staining or retention of lipid.     

The effects of glycol methacrylate as a dehydrating agent on the dimensional changes of liver tissue

The dimensional changes of liver sections during the course of processing with glycol methacrylate (GMA) or with ethanol are described. Tissue processing with ethanol served as a control. During prolonged processing steps (24 h each), linear shrinkage of tissue specimens dehydrated with GMA at room temperature was 13.2%. Subsequent infiltration with GMA resulted in trivial swelling, and polymerization in slight shrinkage (2.3%). In comparison, processing with cold GMA resulted in shrinkage during dehydration (about 10.8%), a slight swelling in pure GMA, followed by shrinkage during polymerization (2.2%). Short routine processing schedules resulted in similar shrinkage/swelling patterns, although precise values differed slightly. In all experiments, ethanolic dehydration resulted in smaller dimensional tissue changes than did GMA dehydration. The dimensional changes of tissue sections during stretching on water, mounting and drying compensated for the major part of the shrinkage manifested during processing.     

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.     

Fluorescence microscopy of rat embryo sections stained with haematoxylin–eosin and Masson's trichrome method

The fluorescence pattern induced by haematoxylin–eosin (HE) and Masson's trichrome (MT) staining methods on paraffin sections of rat embryos (from 13 to 18 days old) has been studied. Using optimal excitation (green light, 545 nm), HE- or MT-stained sections showed a selective red emission of the acidophilic tissue components, which was due to eosin Y in the case of HE and to acid fuchsin and/or xylidine ponceau in the case of MT. The fluorescence intensity induced by these anionic dyes was variable and related to the substrate nature and the embryo age. Whereas in young embryos only the immature red blood cells showed a noticeable fluorescence, in the oldest embryos there were also other tissue components that selectively fluoresced with these dyes, in particular fibre lens cells, elastic fibres, zymogen granules and muscle cells. Spectrofluorometric studies on free dyes and densitometric analysis of protein blots confirmed microscopical observations. Our results indicate that the standard HE and MT staining methods can be used in recognizing the appearance of specific protein structures in embryonic tissues by means of fluorescence microscopy.