Staining with Tetraphenylporphinesulfonate in Vivo

Tetraphenylporphinesulfonate (TPPS), a synthetic, nonnaturally occurring porphine derivative, was administered parenterally to tumor bearing rats and its in vivo localization was studied with fluorescence microscopy. TPPS was selectively localized in elastica and eosinophilic leukocytes, but not in other tissue sites rich in basic protein. The elastica of the aorta and medium sized arteries, as well as elastic fibers of the subendocardium, paratracheal connective tissue and bronchial walls showed the strongest red fluorescence. The intensity of fluorescence in these sites corresponded with the degree of sulfonation of the TPPS. The tumors showed moderate red fluorescence diffusely in the cytoplasm.     

Staining with Tetraphenylporphinesulfonate in Vivo

Tetraphenylporphinesulfonate (TPPS), a synthetic, nonnaturally occurring porphine derivative, was administered parenterally to tumor bearing rats and its in vivo localization was studied with fluorescence microscopy. TPPS was selectively localized in elastica and eosinophilic leukocytes, but not in other tissue sites rich in basic protein. The elastica of the aorta and medium sized arteries, as well as elastic fibers of the subendocardium, paratracheal connective tissue and bronchial walls showed the strongest red fluorescence. The intensity of fluorescence in these sites corresponded with the degree of sulfonation of the TPPS. The tumors showed moderate red fluorescence diffusely in the cytoplasm.     

Fine structure of synapses in the hippocampus of the rabbit with special reference to dark presynaptic endings

Summary The stratum radiatum of h ₃ and h ₄ in the hippocampus of the rahbit, where the mossy fiber endings are distributed, was investigated under the electron microscope. These regions contain a certain number of electron dense presynaptic endings. These are characterized by highly dense synaptic vesicles and mitochondrial matrices. The dense endings are not considered as degenerated. Electron dense silver particles, substituted for zinc, occurred on the synaptic vesicles of these dense terminals as well as the mossy fiber endings after the application of Timm's histochemical method modified for electron microscopy. It is concluded that the dark synaptic endings observed might represent mossy fiber terminals in a special functional phase, or might be the result of structural alteration in the course of tissue preparation. The zinc localized in the synaptic vesicles is thought to be associated with the neurotransmitter present in these endings.     

The structure of the coeliac ganglion of a teleost fish Myoxocephalus scorpius

Summary In order to compare the structure of a teleost sympathetic ganglion with those of other vertebrates, light, fluorescence histochemical and electron microscopy were carried out on the coeliac ganglion of the scorpion fish, Myoxocephalus scorpius. In common with studies on other vertebrates, fluorescence histochemistry distinguished two cell types: a) principal neurones which exhibited low levels of specific catecholamine fluorescence and comprise the majority of neurones in the ganglia, and b) smaller intensely fluorescent cells, some of which had processes tens of micrometers long.With the electron microscope, the principal cells were seen to make axodendritic and axosomatic synapses with axons containing mainly 30 nm agranular vesicles at the synaptic site while in other vertebrates usually only one or other synaptic association is present.Both the somata and the processes of intensely fluorescent cells contain 300–600 nm diameter vesicles many of which have electron dense cores. These cells are also innervated by axons containing 30 nm agranular vesicles.     

The classic Golgi apparatus and vacuoles

The dense vacuoles, considered to be the classic Golgi apparatus in the root meristem ofFagopyrum, were studied by the following methods: 1. Impregnation methods for the demonstration of the Golgi apparatus, 2. cytochemical methods, 3. electron microscopic methods in the light microscope and 4. the electron microscope. A comparison was made with the classic Golgi apparatus in animal cells in the light and electron microscope. Dense vacuoles inFagopyrum and also evidently in other plants, were taken for the classic Golgi apparatus on account of their morphological similarity to the Golgi apparatus in animal cells on impregnation with silver and osmium and their staining preperties with lipoid methods. Dense vacuoles differ from the classic Golgi apparatus in other chemical properties, such as content of phenol substances, etc. No formations were found in animal cells which were similar to dense vacuoles on investigating by electron microscopy. In the electron microscope dense vacuoles have the appearance of derivatives of the normal light vacuoles known in plant cells. They therefore belong to vacuome of plant cell and cannot be analogous to the classic Golgi apparatus in animal cells. Thus the use of the term Golgi apparatus for dense vacuoles is not well founded. A comparison was made of fixation and impregnation used in the light microscope with fixation in the electron microscope. After fixation with permanganate, dense vacuoles have the same shape as after impregnation. After fixation with permanganate, they stain an intense black in the same way as after impregnation with silver and osmium. The form of the vacuoles is dependent on the fixation used. The comparison was made in the light microscope.     

Chromosome banding study of European catfish,Silurus glanis (Pisces,Siluridae)

The karyotype of European catfish (Silurus glanis L.) was analyzed sequentially by means of silver staining and the chromomycin A₃ (CMA₃)/distamycin A (DA)/DAPI fluorescence technique and by C-banding, respectively. The nucleolus organizer regions (NORs) were localized on the submetacentric pair No. 14. Brilliant CMA₃ fluorescent heterochromatin blocks corresponded to the NORs visualized by silver staining. No DA/DAPI-bright positive fluorescent patterns were detected while C-banding led to the detection of specific banding patterns on several chromosome pairs.—Using these banding data, the karyotype of S. glanis was redescribed.     

Pyronin Y as a fluorescent stain for paraffin sections

Pyronin Y has long been used, in combination with other dyes such as Methyl Green, as a differential stain for nucleic acids in paraffin tissue sections. It also forms fluorescent complexes with double-stranded nucleic acids, especially RNA, enabling semi-quantitative analysis of cellular RNA in flow cytometry. However, the possibility of using pyronin Y as a fluorescent stain for paraffin tissue sections has rarely been investigated. We herein report that in sections stained with Methyl Green–pyronin Y, red blood cells, elastic fibre of blood vessels, zymogen granules of pancreatic acinar cells, surface membrane of heptocytes and kidney tubular cells showed strikingly strong green and/or red fluorescence, while the nuclei of cells appeared non-fluorescent. The use of confocal laser-scanning microscope greatly improved the resolution and selectivity of the fluorescent images. Staining with pyronin Y alone gave similar results in terms of fluorescence properties of the specimens. Pretreatment of paraffin sections with RNase significantly reduced cytoplasmic pyronin Y staining as judged by transmission light microscopy, but it had little effect on the fluorescence intensity of red blood cells, elastic fibres and zymogenbreak granules.     

A silver staining method for single-cell gel assay.

The single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains. (J Histochem Cytochem 49:1183-1186, 2001)     

Measurements of DNA damage on silver stained comets using free Internet software

Silver stain offers the possibility to stain comets permanently, but up to now it was impossible to measure the majority of the comet parameters, because the distinction between head and tail was not recognised by software. Here, we report a silver staining protocol that allows the measurement of comet parameters using the free Internet software CASP. We validated the silver stain protocol by comparing the behaviour of the parameter '% DNA in tail' in silver and fluorescent stained comets. The range of % DNA in tail for different visual categories of damage in silver stained comets was similar to that reported with fluorescence staining. The range was for category 0 (no damage), <1%; category 1 (low damage), 1-25%; category 2 (medium damage), >25-45%; category 3 (high damage), >45-70%; category 4 (very high damage), >70%. The mean of % DNA in tail in silver stained comets was also similar to that reported with fluorescence staining. The mean was for category 0, 0.4+/-0.34%; category 1, 12+/-7%; category 2, 37+/-4%; category 3, 57+/-5% and category 4, 83+/-6%. Others comet parameters such as tail length, tail moment and Olive tail moment can be also measured. The silver staining protocol reported here opens new opportunities for those working in the assay without fluorescent microscope as the measurement of comet parameters using free Internet software and conventional microscope becomes possible.     

Immunocytochemical localization of nuclear antigens in the unicellular green alga,Chlamydomonas reinhardtii,processed by cryofixation and freeze-substitution

Summary We describe the preparation of monoclonal antibodies to nuclear antigens in the green alga,Chlamydomonas reinhardtii, and their localization at the light and electron microscope level. Supernatants from hybridomas were screened by the ELISA method and the four antibodies giving the strongest signal were subjected to further analysis. At the LM level immunogold silver staining was used on semi-thick resinless sections. We have examined at the EM level the distribution of these antigens by post-embedding immunocytochemical techniques on sections of conventionally fixed specimens compared to cryofixed and freeze-substituted ones. Enhanced ultrastructural preservation was observed in cells which were cryofixed, freeze-substituted and embedded at –35°C in Lowicryl K4M. Different preparative procedures involving cryofixation and substitution are described. Of the four antibodies three were localized under light and electron microscopy. All three were distributed in the interchromatin space. One of these antigens (QUL4D2, 54 kDa) is also found in the dense fibrillar component and fibrillar centers of the nucleolus.Abbreviations DFC dense fibrillar component - EM electron microscope - FC fibrillar center - GAM5 goat anti-mouse IgM coupled to 5 nm colloidal gold - Ig immunoglobulin - LM light microscope - MAb monoclonal antibody - PAG protein A-gold - PBS phosphate buffered saline - PEG polyethylene glycol     

ULTRASTRUCTURAL LOCALIZATION OF ARGYROPHILIC PROTEINS IN NUCLEOLI OF ZEA MAYS BY TWO SILVER STAINING TECHNIQUES

Ag staining was applied on interphasic nucleoli of Zea mays root cells 120h after germination. We applied the two-step Ag-NOR staining technique to small root fragments and the one-step technique to sections of Lowicryl-embedded tissue. The small-sized silver grains were mainly located in the dense fibrillar component (DFC). The unstained fibrillar centers (FCs) differed in their proteinic contents from the NOR (which is positively silver stained) and were not the interphasic NOR counterpart.     

A promising injectable scaffold: The biocompatibility and effect on osteogenic differentiation of mesenchymal stem cells

Chitosan/β-glycerophosphate/collagen (C/GP/Co) is a promising injectable scaffold in the bone tissue engineering. In this study, we prepared this scaffold and evaluated its biocompatibility and effects on the osteogenic differentiation of mesenchymal stem cells (MSCs). After fabrication, the C/GP/Co hydrogel was examined in a scanning electron microscope (SEM) and showed a porous microstructure. Its biocompatibility was assessed by cell morphology and cell viability assays. Cell morphological observations were performed by fluorescent microscope in 2D cultivation and by laser confocal scanning microscope (LCSM) in 3D cultivation, respectively. Cell viability in 2D and that in 3D cultivation were both evaluated by the Cell Counting Kit-8 (CCK-8) assay. Its effect on osteogenic differentiation of MSCs in vitro was clarified by alkaline phosphatase (ALP) activity, Alizarin Red staining, and real-time polymerase chain reaction (Real-time PCR). An additional experiment of the ectopic bone formation in nude mice was conducted to investigate its effects on osteogenic differentiation of MSCs after subcutaneous injection. The results proved that C/GP/Co hydrogel exhibited good biocompatibility and enhanced the in vitro osteogenic differentiation of MSCs. In the experiment of ectopic bone formation, this hydrogel demonstrated its capability of supporting neovascularization and differentiation of MSCs toward osteogenic lineage. Therefore, C/GP/Co hydrogel scaffold holds a great promise for the bone tissue engineering applications.     

Electron microscopic ammonical silver reaction forTradescantia pollen grain

Summary The postformalin ammonical silver reaction (ASR) for basic nuclear proteins was applied to both the generative and vegetative nuclei ofTradescantia pollen grains and examined with the electron microscope. The ASR deposit was usually observed mainly in the dense chromatic regions of both the generative and vegetative nuclei. Since a large amount of dense chromatic regions was observed in the generative nucleus than the vegetative ones in late or mature pollen grains, as a results, the increased amount of the ASR deposit was revealed in the generative nucleus.     

灰葡萄孢菌过氧化物酶体及细胞核的荧光蛋白标记

[目的]对灰葡萄孢菌(Botrytis cinerea)的细胞核和过氧化物酶体进行荧光蛋白标记,为研究其生长发育和侵染过程中细胞结构和细胞器动态提供基础.[方法]以绿色荧光蛋白(GFP)和红色荧光蛋白(DsRED、mCherry)为报告基因,利用根癌农杆菌介导转化(Agrobacterium tumefaciens m...     

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce (Picea abies (L.) H. Karsten) and silver birch (Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-m cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.Abbreviation CLSM confocal laser-scanning fluorescence microscopy     

Fluorescence microscopic distinction between elastin and collagen

Summary Distinction between elastin and collagen in arteriosclerotic lesions is difficult because immature and incompletely cross-linked collagen bind so-called elastica stains; furthermore, abnormal collagen can lack cross-striation and thus resemble elastin in electron microscopy. However, collagen and elastin differ significantly in their content of basic amino acids and hence in their affinity for heteropolyacids. This chemical difference was utilized for the development of a fluorescence microscopic method for distinction between collagen and elastin.Paraffin sections of human autopsy material were treated with a 1% aqueous solution of phosphomolybdic acid (PMA) for five minutes, rinsed in distilled water, dehydrated and mounted. Other series were treated with the PMA-molybdenum blue reaction and with various special stains.Only elastic membranes of aorta, the elastica interna and externa of sizable arteries, and true elastic fibers remained strongly fluorescent; the autofluorescence of collagen, reticulum fibers, basement membranes, pseudo-elastic fibers, and elastic membranes in small arteries was quenched. In other series PMA abolished the fluorescence of basic fluorochromes.Correlation of fluorescence and direct light microscopic observations with chemical and electron microscopic data showed that the PMA-fluorescence method permits distinction between elastin and various types of collagen.     

Cloning of AFLP markers detected by fluorescence capillary electrophoresis

The available methods to isolate specific amplified fragment length polymorphism (AFLP) markers can be used only if markers are detected by radioactive labeling, silver staining, or ethidium bromide staining; these methods are useless if modern and automated genetic analyzers are used to detect AFLP markers by fluorescent labeling. We have developed a method that allows for isolation and cloning of specific AFLP markers obtained with a laser-induced fluorescence capillary electrophoresis system. This procedure has been tested on 5Arabidopsis thaliana polymorphic AFLP markers, and the nucleotide sequences obtained from these cloned markers were identified and located in theArabidopsis genome.     

Porphyrin cytochrome c. pH effects and interaction with cytochrome-c oxidase

1. Porphyrin cytochrome c, the iron-free derivative of cytochrome c, has been used extensively as a fluorescent analog of cytochrome c. It appears as though its fluorescence intensity but not its relative quantum yield is affected by pH in the physiological range; an apparent pK of about 6.2 is found suggesting a histidine close to the porphyrin. 2. The fluorescence intensity of the porphyrin cytochrome c in the presence of cytochrome c oxidase is independent of pH; this suggests that the oxidase has the capacity to control the pK of whichever group is responsible for the pH sensitivity of the free porphyrin cytochrome c. The most likely candidate for this pH-sensitive group is histidine-18. The N-3 nitrogen of this residue forms one of the axial ligands to the iron in the intact cytochrome c but it is uncoordinated in the iron-free derivative.     

Fluorescent colloidal gold: A cytochemical marker for fluorescent and electron microscopy

Summary The gold method was further developed for fluorescent microscopy. Gold granules (12 nm in size) were labelled with rhodamine conjugates of Concanavalin A and avidin. The fluorescent markers were used to mark cell wall mannan on the yeast Saccharomyces cerevisiae either by the one-step, or by the two-step method via a biotinyl derivative of ConA. By fluorescence or transmission electron microscopy, the two-step method was found to achieve a higher density of marking.     

Fine structure and fluorescence morphology of adrenergic nerves after 6-hydroxydopamine in vivo and in vitro

Summary In parallel fine structural, fluorescence histochemical and biochemical experiments the effect of 6-OH-DA administered in vivo and in vitro on the adrenergic nerves in the mouse iris was studied. As seen in the electron microscope, in vivo administration of 6-OH-DA causes a selective, rapid degeneration of the adrenergic axon terminals similar to that found after axotomy, whereas the cholinergic nerves are unaffected at all time intervals studied. Already 1 hr after the injection of 6-OH-DA the axonal enlargements swell and the size of the dense core of the granular vesicles is strongly reduced. Since the NA stores are almost completely depleted at this time interval, the small core present may be due to a reaction between 6-OH-DA and the fixative. From 2–4 hr after the injection increasing numbers of axonal enlargements with a high electron density are observed in the Schwann cell cytoplasm, which later are digested and completely absent about 48–72 hr after the 6-OH-DA injection. During the following weeks adrenergic axons reappear. This time course of degeneration obtained is considerably faster than that seen after axotomy in other studies. After incubation in 6-OH-DA containing media similar changes were observed in the axonal enlargements, starting already after 30 min of incubation. At this time-point there is a considerable reduction of endogenous NA and a severe damage of the membrane pump uptake mechanism. Incubation with 6-OH-DA and subsequent rinsing for 2 hr caused marked changes, including partly swelling of axons and partly shrinking of the axons into electron dense bodies.The fluorescence histochemical and biochemical results are in good agreement with the ultrastructural studies demonstrating a rapid loss of NA from the adrenergic nerve terminals and main axons and a long lasting depletion of the NA, with a gradual recovery to 75% 6 weeks after the injection.The investigation has been supported by research grants from the Swedish Medical Research Council (14X-2295, 14X-2887 and 04X-3881) Karolinska Institutet, Magnus Bergvalls and Carl-Berthel Nathorst Stiftelser. For generous supplies of drugs we are indebted to the following companies: AB Hässle (6-OH-DA, through Dr H. Corrodi), Pfizer (Niamid^®), Ciba (Serpasil^®). The skilful technical assistance of Miss Bodil Flock, Mrs Waltraut Hiort and Mrs Eva Lindqvist is gratefully acknowledged.