A method for the staining of intraosseous nerve fibers using Sihler’s staining technique

Abstract

Understanding nerve fiber distribution in the jaw bone is important when performing invasive surgical treatments. Both microscopic and macroscopic anatomical techniques have been developed to study innervation. Conventional methods of removing and staining these structures, however, often alter structure and lack reproducibility of the resulting specimens. We sought to optimize Sihler’s staining technique to stain intraosseous nerves in mandibles. Four cadaver specimens were used. The best staining of intraosseous nerve fibers was achieved by using the Plank-Rychlo solution. When the Styrene monomer was used, the resulting transparency was better than that obtained with glycerin under the same conditions. No significant differences were found between Sihler's staining procedure performed according to the conventional method and the procedure in which the second decalcification step was omitted. Our results demonstrate that applying Sihler’s staining technique to bones makes them transparent and allows observation of nerves while preserving the external shape of the bone and maintaining the position of intraosseous nerve fibers. Our findings suggest our Sihler staining method for intraosseous nerve fibers can provide an intermediate resolution between macroscopic and microscopic techniques.     

Effects of α-galactosidase digestion on lectin staining in human pancreas

Summary Effects of -galactosidase (from green coffee beans) digestion on lectin staining were examined in formalin-fixed, paraffin-embedded human pancreatic tissues from individuals of blood-group B and AB. Digestion with the enzyme resulted in almost complete loss of Griffonia simplicifolia agglutinin I-B₄(GSAI-B₄) staining in the acinar cells with concomitant appearance of Ulex europaeus agglutinin-I(UEA-I) staining in the corresponding cells. In addition, reactivity with soybean agglutinin(SBA) was also imparted by the enzyme digestion in GSAI-B₄ positive acinar cells. -Galactosidase digestion following -galactosidase digestion neither reduced the reactivity with SBA nor induced the reactivity with Griffonia simplicifolia agglutinin-II(GSA-II) in GSAI-B₄ positive cells, while in UEA-I positive cells, both reduction of SBA reactivity and appearance of GSA-II reactivity occurred after simple -galactosidase digestion as well as sequential digestion with - and -galactosidase. However, when -l-fucosidase digestion procedure was inserted between - and -galactosidase digestion, UEA-I staining imparted by -galactosidase digestion was markedly decreased in intensity and GSA-II reactivity was appeared in GSAI-B₄ positive acinar cells. Furthermore, after sequential digestion with -galactosidase and fucosidase, reactivity with peanut agglutinin(PNA) was revealed in GSAI-B₄ positive acinar cells as well as UEA-I positive cells in secretors. In non-secretors, strong PNA staining was usually observed in the acinar cells throughout the glands without enzyme digestion. These results confirmed that the -galactosidase induced GSA-II reactivity and the fucosidase induced PNA reactivity are due to precursors of different kinds of blood-group determinants and suggest that at least two kinds of B antigen determinants, i.e. Gal(1-3)[Fuc(1-2)]Gal(1-3,4)GlcNac and Gal(1-3)-[Fuc(1-2)]Gal(1-3)GalNAc are produced in GSAI-B₄ positive acinar cells. The synthesis of the latter type of B antigen is assumed to be controlled under the secretory gene in human pancreas.Abbreviation GalNAc N-acetyl-d-galactosamine - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Fuc l-fucose - NeuNAc N-acetylneuraminic acid (sialic acid)     

A study of staining for electron microscopy using collagen as a model system—V. The effect of suberimidate fixation on negative staining patterns

The effects of the fixative dimethylsuberimidate (DMS) on negative staining patterns were studied using reconstituted fibrils of type I calf skin collagen (of known amino acid sequence) as a model system and comparing electron-optical data and chemical data by a computer-aided correlation procedure. The results show that the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains is the dominant factor in determining the stain-excluding property of a DMS-fixed negatively stained collagen fibril as it is in unfixed collagen. Some contribution of positive staining can also be demonstrated after DMS-fixation by partial correlation analysis. Other evidence suggests that (unlike glutaraldehyde) DMS does not produce any morphological alterations to the negative staining pattern.     

Peroxisomes: 40 years of histochemical staining,personal reminiscences

The historical circumstances that led to the discovery of the 3,3′-diamino-benzidine (DAB) method for staining of peroxisomes 40 years ago are reviewed. In the course of studies on the uptake and absorption of horse radish peroxidase in mammalian liver, in sections incubated for detection of peroxidase activity in DAB, it was noted that peroxisomes also stained positively for peroxidase activity. Subsequently, it was revealed that the peroxidatic activity of catalase, which is abundantly present in peroxisomes, is responsible for that staining. This notion was confirmed in quantitative biochemical studies with crystalline beef liver catalase and in tracer studies using catalase as an ultrastructural tracer. The application of the DAB method led to the discovery of peroxisomes as a ubiquitous eukaryotic cell organelle, attracting great interest in their investigation in biomedical research.     

Improved in situ β-galactosidase staining for histological analysis of transgenic mice

The present study describes a novel method for the histochemical demonstration of -galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) with 5-bromoindolyl--o-galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial -galactosidase (lacZ). After -galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo-Gal results in greatly enhanced sensitivity of the staining method for -galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of -galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter.     

Technical aspects of the use of 3′,6′-diacetyl fluorescein for vital fluorescent staining of bacteria

Viable bacteria of several species have the capability to incorporate 3′,6′-diacetyl fluorescein (FDA) and rapidly hydrolyze it to fluorescein, which is stored intracellularly. However, several strains of viableEscherichia coli andAlcaligenes faecalis do not evolve and accumulate significant amounts of fluorescein when incubated on glass slides in the presence of FDA. In the present study, 10⁵–10⁷ E. coli orA. faecalis bacteria (viability more than 95%) were accumulated in separate experiments on 0.45-μm membrane filters and then stained for 5–10 min with FDA diluted immediately before use in phosphate-buffered saline, freshly prepared nutrient broth, or nutrient broth preconditioned by overnight growth of the respective bacteria. It was shown that in all cases about 20% of the bacteria did evolve significant amounts of fluorescein, thus enabling a visual observation of these cells in the fluorescence microscope.Bacillus cereus bacteria—that evolved and accumulated fluorescein on glass slides—were shown to be fluorescent on membrane filters after FDA staining. 100%, 40%, or 70% of the bacteria were stained if the FDA solution used had been prepared in nutrient broth preconditioned by overnight growth of the same bacteria, fresh nutrient broth, or phosphate-buffered saline, respectively. This preliminary study indicates the necessity of determining the technical conditions required for FDA staining for each bacterial species under study.     

Is beta-galactosidase staining a marker of senescence in vitro and in vivo?

Cytochemically detectable beta-galactosidase (beta-gal) at pH 6.0 has been reported to increase during the replicative senescence of fibroblast cultures and has been used widely as a marker of cellular senescence in vivo and in vitro. In this study, we have characterized changes in senescence-associated (SA) beta-gal staining in early and late passage cultures, cultures established from donors of different ages, virally immortalized cells, and tissue slices obtained from donors of different ages. The effects of different culture conditions were also examined. While we confirm the previous report that SA beta-gal staining increased in low-density cultures of proliferatively senescent cells, we were unable to demonstrate that it is a specific marker for aging in vitro. Cultures established from donors of different ages stained for SA beta-gal activity as a function of in vitro replicative age, not donor age. We also failed to observe any differences in SA beta-gal staining in skin cells in situ as a marker of aging in vivo. The level of cytochemically detectable SA beta-gal was elevated in confluent nontransformed fibroblast cultures, in immortal fibroblast cultures that had reached a high cell density, and in low-density, young, normal cultures oxidatively challenged by treatment with H2O2. Although we clearly demonstrate that SA beta-gal staining in cells is increased under a variety of different conditions, the interpretation of increased staining remains unclear, as does the question of whether the same mechanisms are responsible for the increased SA beta-gal staining observed in senescent cells and changes observed in cells under other conditions.     

Limitations of safranin ‘O’ staining in proteoglycan-depleted cartilage demonstrated with monoclonal antibodies

Summary The intensity of safranin O staining is directly proportional to the proteoglycan content in normal cartilage. Safranin O has thus been used to demonstrate any changes that occur in articular disease. In this study, staining patterns obtained using monoclonal antibodies against the major components of cartilage proteoglycan chondroitin sulphate (anti CS) and keratan sulphate (anti KS), have been compared with those obtained with safranin O staining, in both normal and arthritic tissues. In cartilage where safranin O staining was not detectable, the monoclonal antibodies revealed the presence of both keratan and chondroitin sulphate. Thus, safranin O is not a sensitive indicator of proteoglycan content in diseases where glycosaminoglaycan loss from cartilage has been severe.     

A study of staining for electron microscopy using collagen as a model system—VI. Uranyl nitrate negative staining patterns and their correlation with sequence data

Collagen is used as a model system to study the mechanism of negative staining. Negative staining patterns from reconstituted fibrils of type I calf skin collagen (of known amino acid sequence) were compared with chemical data by a computer-aided correlation procedure. The stain used was uranyl nitrate, pH 3.2 and 4.9. The results show that the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains is the dominant stain-excluding factor determining the small-scale distribution of stain along the collagen fibril. Some contribution of positive staining can also be demonstrated by the analysis described here.     

Ergosterol — A measure of fungal growth in wood for staining and pitch control fungi

Summary A modified ergosterol analysis method, including a simultaneous saponification and refluxing extraction procedure along with HPLC quantification, was used to measure fungal colonization rate in wood. In liquid media the ergosterol content of the mycelia was measured and correlated to the fungal dry weight. In work on investigating staining fungal proteinase production on wood, ergosterol values were used to monitor fungal growth and to determine when maximum proteinase activity occurred. Similarly, we correlated ergosterol values with the decrease of wood lipids during pitch control fungal colonization.     

Reliability of cartilage digestion and FDA–EB fluorescence staining for the detection of chondrocyte viability in osteochondral grafts

The purpose of this study is to evaluate the reliability of cartilage digestion and fluorescein diacetate-ethidium bromide (FDA–EB) fluorescence staining for the detection of chondrocyte viability in osteochondral grafts. Sixteen fresh osteochondral grafts were harvested from pig knee condyles, and the articular cartilage tissue was preserved. Each cartilage graft was cut into two 70-µm thick pieces and randomly allocated to Group A or Group B. The cell viability of Group A was detected using FDA–EB fluorescence staining of the digested cartilage, and the viability of Group B was detected with FDA–EB fluorescence staining of cartilage sections. Comparisons of chondrocyte viability and correlation analyses of the two groups were performed using the paired sample t test and Pearson correlation test, respectively. No significant difference was found in the chondrocyte viability between Groups A and B (p > 0.05), and a strong correlation was observed (r = 0.70, p < 0.05). Therefore, cartilage digestion with FDA–EB fluorescence staining is a reliable method for detecting chondrocyte viability in osteochondral grafts.     

Ultrastructure of Sarcocystis tenella. I. Endozo?te (after negative staining]

The employment of negative staining technics for the endozoites (cyst stages) of Sarcocystis tenella allowed the elucidation of certain aspects of their fine structure. The conoid consists of similar to 20 oblique fibers and is surmounted by a ring with regular ornamentation. In the conoid's interior there are 2 excentric parallel microtubules which extend posteriorly for a considerable distance into the adjacent cytoplasm. The fibers of the conoid, intraconoid microtubules, appear to have the same diameter and structure as the 22 subpellicular microtubules. They are "cemented" anteriorly into a periconoidal ring which surrounds the conoid. The "reticulated" pellicle has certain differentiations: the micropore, surrounded by a "fibrillar" element, similar to 10 subcircular structures arranged into an anterior crown, and 11 rows of granules converging toward the posterior end. The sarconemes look like rice grains which, contrary to previous statements, are independent of one another. It is established that there are only 2 rhoptries.     

A study of staining for electron microscopy using collagen as a model system—IV. Phosphotungstate/tungstate negative staining patterns and their correlation with sequence data

The type I collagen fibril (for which the axial distribution of amino acid residues is known) has been used as a model system to study the mechanism of negative staining. Negative staining patterns from reconstituted fibrils were compared with chemical data by a computer-aided correlation procedure. Stains used were: phosphotungstic acid, pH 3.2 and 7.0, lithium tungstate, pH 7.2, and methylamine tungstate, pH 6.6. In all cases, the results of the correlation analyses point to the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains as the dominant stain-excluding factor determining the small-scale distribution of stain along the collagen fibril. Some preferred uptake of heavy metal ions on charged side chains (a positive staining contribution) can be demonstrated by partial correlation analysis but, under the staining conditions used here, the effect is largely masked by the much greater negative staining component.     

Novel fluorescence probes based on the chalcone scaffold for in vitro staining of β-amyloid plaques

The β-amyloid (Aβ) plaque is one of the neuropathological hallmarks in the Alzheimer’s disease brain. The detection of Aβ plaques with fluorescence probes is useful for preclinical studies of Alzheimer’s disease. In this study, we developed four novel fluorescence probes based on chalcone scaffold. In an in vitro binding study, all FCH derivatives showed moderate binding affinity for Aβ(1–42) aggregates (K_i?=?72–114?nM). The fluorescence intensities of FCH-3 and FCH-4 dramatically changed in the presence of Aβ(1–42) aggregates (6.7 and 14.2 fold), but the changes of FCH-1 and FCH-2 were minor (2.0 and 2.4 fold). In a fluorescence staining study using Tg2576 mouse brain sections, FCH-3 and FCH-4 clearly visualized Aβ plaques, but FCH-1 and FCH-2 did not stain. Taken together, all FCH derivatives could bind to Aβ aggregates, but only FCH-3 and FCH-4 may be useful fluorescence probes for in vitro staining of Aβ plaques.     

Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by β-mercaptoethanol for specific staining of tetracysteine-tagged proteins

Recent advances in the field of small molecule labels for live cell imaging promise to overcome some of the limitations set by the size of fluorescent proteins. We tested the tetracysteine–biarsenical labeling system in live cell fluorescence microscopy of reggie-1/flotillin-2 in HeLa and N2a cells. In both cell types, the biarsenical staining reagent FlAsH/Lumio Green accumulated in active mitochondria and led to mitochondrial swelling. This is indicative of toxic side effects caused by arsenic, which should be considered when this labeling system is to be used in live cell imaging. Mitochondrial accumulation of FlAsH/Lumio Green was reversed by addition of low concentrations of thiol-containing reagents during labeling and a subsequent high stringency thiol wash. Both ethanedithiol and β-mercaptoethanol proved to be effective. We therefore established a staining protocol using β-mercaptoethanol as thiol binding site competitor resulting in a specific staining of tetracysteine-tagged reggie-1/flotillin-2 of adequate signal to noise ratio, so that the more toxic and inconvenient ethanedithiol could be avoided. Furthermore, we show that staining efficiency was greatly enhanced by introducing a second tetracysteine sequence in tandem.M.F. Langhorst and S. Genisyuerek contributed equally to this work.     

A study of staining for electron microscopy using collagen as a model system—VII. The effect of formaldehyde fixation

Exposure to formaldehyde brings about small but readily detectable changes in the staining behaviour of collagen fibrils. These changes can be interpreted in chemical terms by comparing fibril staining patterns with artificial patterns computer-generated from sequence data. Positive staining with phosphotung-state (where heavy metal is confined to anions), shows that most of the lysyl and hydroxylysyl side-chains lose their charge character as a result of formaldehyde treatment and cease to take up staining ions. The charge character of arginyl (and probably histidyl) residues is unaltered and these residues continue to react with stain. Acidic residues are also unaffected. These results accord with biochemical evidence that the initial reaction between proteins and formaldehyde leading to subsequent cross-linking involves modification of ε-amino (and α-amino) groups. They show too that the secondary condensation producing the actual cross-link does not alter the charge character of the second group, at least when it is on an arginyl (or histidyl) side-chain.Formaldehyde-induced changes in stain deposition can also be detected after negative staining, although they are slight compared with those brought about by glutaraldehyde. Unlike glutaraldehyde, formaldehyde introduces no bulky polymeric adducts into the fibril structure, and the conspicuous stain-excluding bands seen in negative staining patterns following glutaraldehyde fixation are absent after exposure to formaldehyde. For this reason, where chemical fixation is used to stabilize macromolecules and supramolecular aggregates prior to negative staining and high resolution electron optical imaging, formaldehyde would seem to be preferable to glutaraldehyde. Data from fibril staining patterns and from thermal stability measurements (made on collagen gels) show that formaldehyde fixation does not preclude a subsequent reaction with glutaraldehyde.As with other fixatives, there is reduced accessibility to stain after formaldehyde treatment. Accessibility is least in the overlap zone where the denser packing of collagen molecules provides greater opportunities for intermolecular cross-linking. Gel electrophoresis confirms that formaldehyde-induced cross-links in fibrils are predominantly intermolecular.     

A negative staining study of natural and synthetic L-α-lysophosphatidylcholine micelles,macromolecular aggregates and crystals

A comparative assessment has been made by transmission electron microscopy of negatively stained specimens, of the micellar, aggregated and crystalline states of palmitoyl, oleoyl and ex ovo L-α-lysophosphatidylcholine present in aqueous suspensions. Micelle formation from dry lysophosphatidylcholines is shown to be temperature dependent. The presence of the unsaturated fatty acid in oleoyl L-α-lysophosphatidylcholine and some degree of unsaturation in L-α-lysophosphatidylcholine (ex ovo) promotes micelle formation at low temperatures (4 and 22°C), whereas crystalline palmitoyl L-α-lysophosphatidylcholine is essentially insoluble at low temperatures and requires incubation at 60°C to produce a micellar suspension.It is suggested that the micellar conformation is not spherical, a cylindrical or discoid shape is more compatible with the images presented. Both palmitoyl and ex ovo L-α-lysophosphatidylcholine produce flexibel rod-like micellar aggregates ca 6 nm in diameter and larger (20–60 nm dia) stacked-disc aggregates, again with a temperature dependency. The thickness of the disc-like L-α-lysophosphatidylcholine of a phospholipid bilayer (ca 6–7 nm). This, together with the ability of palmitoyl L-α-lysophosphatidylcholine to crystallize as multi-lamellar hexagonal particles which remain stable in aqueous suspensions at 4°C, suggests that, as with other phospholipids, the L-α-lysophosphatidylcholines possess the property of forming lamellar structures, but that these become increasingly unstable at higher temperatures depending on the fatty acid unsaturation. Ammonium molybdate and sodium phosphotungstate have been found to be more satisfactory than uranyl acetate for negative staining of aqueous suspensions of L-α-lysophosphatidylcholines.     

Histochemische Reaktion und „negative staining“ von Zellfraktionen

Zusammenfassung Ein mit Formvar-Kohle befilmtes Netz wird auf die Oberfläche einer suspendierten Zellfraktion gelegt. Ein Teil der Suspension wird somit aufgenommen. Für die Untersuchung von morphologischen Strukturen wird es sofort auf die Oberfläche einer Waschlösung übertragen, zur Identifizierung von histochemischen Zellstrukturen auf eine Inkubationslösung. Danach wird das Netz auf die Oberfläche der negative-staining-Lösung (PWS 10%, pH 6,5 mit 1 n NaOH) gebracht und anschließend nochmals gewaschen. Morphologische und histochemische (Säurephosphatase nach Gomori) Befunde an Membranen und Partikeln einer Fraktion der Rattenmilz werden diskutiert.

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Summary A formvar-carbon coated grid is placed on the surface of a suspension of a cell fraction. Part of the cellular material will adhere to the formvar-carbon film. For morphologic investigation the grid is immediately transferred to the surface of a washing solution; for histochemical identification of cell structures the grid is however placed on the surface of a incubation medium. After washing or incubation, the grid is negatively stained, i.e. transferred to the surface of the PTA solution (PTA 10%, pH=6,5 with 1 n NaOH), and finally again washed for a short time. Morphological and histochemical (Gomori method for the localisation of the acid phosphatase activity) findings on membranes and particles of a fraction of rat spleen are reported.