Chemical Fixation Methods Research Articles

Live-cell imaging and quantitative analysis of embryonic epithelial cells in Xenopus laevis.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues(1). A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Chemical fixation of polyelectrolyte multilayers on polymer substrates

A simple chemical fixation method for the fabrication of layer-by-layer (LbL) polyelectrolyte multilayer (PEM) has been developed to create a large area, highly uniform film for various applications. PEM of weak polyelectrolytes, i.e., polyallylamine hydrogen chloride (PAH) and poly(acrylic acid)(PAA), was assembled on polymer substrates such as poly(methyl methacrylate)(PMMA) and polycarbonate (PC). In the case of a weak polyelectrolyte, the fabricated thin film thickness of the polyelectrolyte multilayers was strongly dependent on the pH of the processing solution, which enabled the film thickness or optical properties to be controlled. On the other hand, the environmental stability for device application was poor. In this study, we utilized the chemical fixation method using glutaraldehyde (GA)-amine reaction in order to stabilize the polyelectrolyte multilayers. By simple treatment of GA on the PEM film, the inherent morphology was fixed and the adhesion and mechanical strength were improved. Both surface tension and FT-IR measurements supported the chemical cross-linking reaction. The surface property of the polyelectrolyte films was altered and converted from hydrophilic to hydrophobic by chemical modification. The possible application to antireflection coating on PMMA and PC was demonstrated.

In situ chemical fixation of arsenic-contaminated soils: An experimental study

This paper reports the results of an experimental study testing a low-cost in situ chemical fixation method designed to reclaim arsenic-contaminated subsurface soils. Subsurface soils from several industrial sites in southeastern U.S. were contaminated with arsenic through heavy application of herbicide containing arsenic trioxide. The mean concentrations of environmentally available arsenic in soils collected from the two study sites, FW and BH, are 325 mg/kg and 900 mg/kg, respectively. The soils are sandy loams with varying mineralogical and organic contents. The previous study [Yang L, Donahoe RJ. The form, distribution and mobility of arsenic in soils contaminated by arsenic trioxide, at sites in Southeast USA. Appl Geochem 2007;22:320-341] indicated that a large portion of the arsenic in both soils is associated with amorphous aluminum and iron oxyhydroxides and shows very slow release against leaching by synthetic precipitation. The soil's amorphous aluminum and iron oxyhydroxides content was found to have the most significant effect on its ability to retain arsenic. Based on this observation, contaminated soils were reacted with different treatment solutions in an effort to promote the formation of insoluble arsenic-bearing phases and thereby decrease the leachability of arsenic. Ferrous sulfate, potassium permanganate and calcium carbonate were used as the reagents for the chemical fixation solutions evaluated in three sets of batch experiments: (1) FeSO(4); (2) FeSO(4) and KMnO(4); (3) FeSO(4), KMnO(4) and CaCO(3). The optimum treatment solutions for each soil were identified based on the mobility of arsenic during sequential leaching of treated and untreated soils using the fluids described in EPA Method 1311 [USEPA. Method 1311: toxicity characteristic leaching procedure. Test methods for evaluating solid waste, physical/chemical methods. 3rd ed. Washington, DC: U.S. Environmental Protection Agency, Office of Solid Waste. U.S. Government Printing Office; 1992] toxic characteristics leaching procedure (TCLP) and EPA Method 1312 [USEPA. Method 1312: synthetic precipitation leaching procedure. Test methods for evaluating solid waste, physical/chemical methods. 3rd ed. Washington, DC: U.S. Environmental Protection Agency, Office of Solid Waste. U.S. Government Printing Office; 1994] synthetic precipitation leaching procedure (SPLP). Both FW and BH soils showed significant decreases in arsenic leachability for all three treatment solutions, compared to untreated soil. While soils treated with solution (3) showed the best results with subsequent TCLP sequential leaching, SPLP sequential leaching of treated soils indicated that lowest arsenic mobility was obtained using treatment solution (1). Treatment solution (1) with only FeSO(4) is considered the best choice for remediation of arsenic-contaminated soil because SPLP sequential leaching better simulates natural weathering. Analysis of treated soils produced no evidence of newly-formed arsenic-bearing phases in either soil after treatment. Sequential chemical extractions of treated soils indicate that surface complexation of arsenic on ferric hydroxide is the major mechanism for the fixation process.

High Pressure Freezing (HPF)을 이용한 조류 Ptilota filicina의 미세구조 관찰:HPF 고정법과 화학 고정법의 비교

In preparation of the biological samples for electron microscopy, the chemical fixation by glutaraldehyde, paraformaldehyde, and OsO4 has been generally used for a long time. However, the chemical fixation method has some problems: the infiltration time is a little bit long and the ultrastructure of cell or tissue transforms before complete fixation of sample. So, recently, cryo-fixation is considered more often in biomedical field. In this study, we compared High Pressure Freezing (HPF) method with chemical fixation method using a algal sample (Ptilota filicina J. Agardh), which was difficult to fix using chemical fixation method. In chloroplast, the ultrastructure of thylakoid lamella and phycobilisome can not show clearly by chemical fixation. In this study we could observe the ultrastructure of thylakoid lamella and phycobilisome of chloroplast very clearly using HPF fixation. An improved images of ultrastructures of nucleus, mitochondrion and floridean starch could obtain. These results suggest that HPF method is very useful method in algal specimen for electron microscopy.

Immunocytochemical identification of interstitial cells of Cajal in the murine fundus using a live-labelling technique

Interstitial cells of Cajal (ICC) within the gastrointestinal (GI) tract play a critical role in the generation of electrical slow waves and as mediators of enteric motor neurotransmission. Kit immunohistochemistry has proven to be a reliable method to identify the location of these cells within the tunica muscularis and to provide information on how the distribution and density of these cells change in a variety of GI motility disorders. Because of the labile nature of Kit or its detection, ultrastructural immunocytochemistry using conventional chemical fixation methods has been difficult. We describe a novel in vivo technique to label ICC within GI tissues. Using antibodies directed against the extracellular domain of the Kit receptor, we have been able to live-label the stomach with Kit while the animal is under anaesthesia and the organ is still receiving normal blood supply. This approach provided optimum maintenance of ultrastructural features with significant binding of antibody to the Kit receptor. The loss of ICC in many human motility disorders suggests exciting new hypotheses for their aetiology. This method will prove useful to investigate the ultrastructural changes that occur in ICC networks in animal models of motility disorders that are associated with the loss of these cells.

Application of in vivo cryotechnique to the examination of cells and tissues in living animal organs.

When all biological materials in cells and tissues of living animal organs are quickly and promptly frozen, immunolocalization of their components and structural features in situ is necessary to understand their in vivo functioning states. However, these direct morphological analyses were difficult to achieve by conventional chemical fixation methods during the last century. A new cryofixation method, named the "in vivo cryotechnique", in which the normal blood circulation in living animals is always retained at the moment of freezing, has become a powerful tool to visualize the real native morphology of cells and tissues with functional meaning. The "in vivo cryotechnique" can usually be combined with a wide range of subsequent preparation techniques, and can thereby enable us to perform various direct analyses on biological samples, reflecting the physiological functions of living animal organs.

Ultrastructural preservation of biofilms formed by non-typeable Hemophilus influenzae

There is growing evidence to suggest that non-typeable Hemophilus influenzae (NTHi), an important cause of otitis media in children, is able to grow as a biofilm in the middle ear. This observation may help to explain bacterial persistence in chronic infections. We evaluate the usefulness of rapid freezing and freeze substitution as a means of preparing biofilms for ultrastructural examination by comparing the morphology of cryofixed specimens with the morphology produced using more conventional chemical fixation and dehydration methods. Chemical fixation and dehydration methods produced substantial ultrastructural damage to individual NTHi in the biofilm and loss of extracellular matrix, even in the presence of ruthenium red. In comparison, cryofixed and freeze-substituted NTHi biofilms showed significantly improved preservation of bacterial ultrastructure and biofilm organization. The intracellular contents of NTHi prepared using the cryomethods showed little evidence of aggregation, and bacteria within the biofilm were closely packed and surrounded by an abundant extracellular matrix. Although high-pressure freezing of NTHi biofilms followed by freeze substitution was highly effective for preserving ultrastructure when examined by transmission electron microscopy, immersion in liquid propane offered an alternative, “less technical”, freezing method. Immersion in liquid propane followed by freeze substitution and critical point drying was most effective for preserving ultrastructural details in specimens examined by scanning electron microscopy.

Cryo‐sectioning and chemical‐fixing ultramicrotomy techniques for imaging rubber latex particle morphology

Two methods adapted from biological microscopy are described for a new application in imaging the morphology of rubbery latex particles. In the first method, a drop of latex is frozen in liquid nitrogen, sectioned with a diamond knife and vapour-stained with osmium tetroxide, then viewed by transmission electron microscopy. When applied to latexes made by emulsion polymerization of methyl methacrylate in a natural rubber latex seed, inclusions are clearly visible. A chemical fixation method is then described for imaging the morphology of such rubbery latex particles. Glutaraldehyde is added to the latex, followed by osmium tetroxide. The sample is then dehydrated in ethanol, epoxy resin added, and the sample cured, ultramicrotomed, and imaged with transmission electron microscopy. An inclusion morphology is again clearly seen.

An improved chemical fixation method suitable for immunogold localization of green fluorescent protein in the Golgi apparatus of tobacco Bright Yellow (BY-2) cells.

In plant systems, the green fluorescent protein (GFP) is increasingly used as a marker to study dynamics of the secretory apparatus using fluorescence microscopy. The purpose of this study was to immunogold localize the GFP, at the electron microscopic level, in a line of tobacco BY-2-cultured cells, expressing a GFP-tagged Golgi glycosyltransferase. To this end we have developed a simple, one-step chemical fixation method that allow good structural preservation and specific labeling with anti-GFP antibodies. Using this method, we have been able to show that an N-glycan GFP-tagged xylosyltransferase is specifically associated with Golgi stacks of BY-2 transformed cells and is preferentially located in medial cisternae. As an alternative to cryofixation methods, such as high-pressure freezing, which requires specialized and expensive equipment not available in most laboratories, this method offers researchers the opportunity to investigate GFP-tagged proteins of the endomembrane system in tobacco BY-2 cells.

Cytochemistry of gastric parietal cells with high-pressure freezing followed by freeze-substitution.

Gastric parietal cells were examined for changes in their ultrastructure and distribution of the proton pump during feeding and fasting states in rats. The fundic glands from rats fed ad libitum or fasted with free access to water were cryofixed using high-pressure freezing followed by freeze-substitution in acetone containing osmium or acrolein and then embedded in Epon 812 or Lowicryl K4M resin, respectively. Excellent ultrastructural preservation was achieved. During the feeding state, intracellular canaliculi and numerous microvilli were well developed, while tubulovesicles were poorly developed. In contrast, during the fasting state, the microvilli in the narrowed space of the intracellular canaliculi were tightly packed and the tubulovesicles were enlarged. Ultrathin sections were immunostained with antibodies against the alpha- and beta-subunits of the proton pump, H+ x K(+)-ATPase, using the immunogold method. The labelling was strong and clearly localized in comparison with that obtained using the conventional chemical-fixation method. Each subunit was localized on the membrane of the microvilli, intracellular canaliculi and tubulovesicles. The distribution of subunit proteins varied between the two states. During ad libitum feeding, the immunolabelling was localized strongly on the membranes of the microvilli and intracellular canaliculi. In contrast, the labelling was strong on the tubulovesicle membrane in the fasting state. The results obtained with each anti-subunit antibody by H+ x K(+)-ATPase immunostaining revealed differences in distribution and labelling density between the feeding and fasting states.

Evaluation of dendritic cell immunogenicity after activation and chemical fixation: a mixed lymphocyte reaction model.

Dendritic cells (DC) are central to the control of adaptive immunity. Their ability to activate antigen-specific T cells depends on their maturation state. Many microbial and inflammatory products have stimulated DC maturation. This in vitro study used assays of phenotype and function to examine the potential of bacillus Calmette-Guerin, muramyl dipeptide, and CpG-rich oligodeoxynucleotides to stimulate DC maturation. A chemical fixation method was developed to reliably assess the functional potential of stimulated DC within a mixed lymphocyte reaction model. Using this method, it was shown that bacillus Calmette-Guerin provides a maturation signal as effective as the prototype DC stimulant interleukin-1beta. Furthermore, weaker stimuli such as muramyl dipeptide and CpG-rich oligodeoxynucleotides also are able to induce functional maturation of DC. Using chemical fixation, it was possible to generate stable DC in an immature or a mature state. These observations have importance for our understanding of the regulation of adaptive immunity and for the design of DC-based immunotherapeutic strategies.

Cytochemical investigation of gastric gland component cells with high-pressure freezing followed by freeze-substitution and hydrophilic resin embedding.

Gastric gland component cells were electron-microscopically and immunoelectronmicroscopically examined with high-pressure freezing followed by freeze substitution and a low-temperature embedding resin method and compared to that of the conventional chemical-fixation method. The rat gastric gland was high-pressure frozen, freeze-substituted with acetone-containing osmium or acrolein, and embedded in Epon 812 or Lowicryl K4M, respectively. Using the high-pressure freezing method, the vitreous freezing range reached the depth of 150 microns from the surface. The ultrathin sections from both procedures embedding in Epon 812 and Lowicryl K4M were doubly stained with uranyl acetate and lead acetate, and histochemically or immunohistochemically stained, respectively. In comparison to the conventional chemical fixation method, excellent results were obtained with respect to ultrastructural preservation. The stainings performed in this experiment included periodic acid-thiocarbohydrazide-silver proteinate staining, cationic colloidal cold at pH 2.5 staining, Helix pomatia lectin-staining, anti-alpha or -beta subunit antibodies of H+K(+)-ATPase immunostaining and pepsinogen immunostaining. The staining intensity of those was stronger than that of the conventional immersion-chemical fixation method. In addition to these results, the labels also showed good specific localization. In this paper, we provide a description of the high-pressure freezing followed by freeze substitution and low-temperature embedding resin method compared to the conventional chemical-fixation method. Our results suggest that this method is a suitable tool for ultrastructural and histochemical/immunohistochemical studies at high resolution.

Microscopic studies on mature flax fibers embedded in LR white: immunogold localization of cell wall matrix polysaccharides.

Flax fibers have been the subject of many biochemical studies, which revealed that cellulose and pectins are the major constituents of their walls. In contrast, little is known about the location of these polymers within the walls of mature fibers by microscopic methods. This has been technically hampered by the very thick secondary wall of fibers, resulting in inadequate tissue preservation unsuitable for immunogold microscopy. In this study, we adapted the basic chemical fixation, dehydration and infiltration methods to achieve a good preservation of the cell structures of mature fibers and reduced damage to antigens. We were able to apply postembedding immunocytochemical techniques to map the location of various pectic epitopes within the walls of mature fibers. Our immunolabeling data show that homogalacturonans were exclusively found in the middle lamellae and the cell junctions but were not detectable in the secondary wall. In contrast, rhamnogalacturonan I (RG I)-associated epitopes, as well as galactan and arabinan epitopes, were abundantly distributed over the secondary wall of mature fibers.

Trace Metal Detection by X-ray Microanalysis in Vascular Cells of Roots and Leaves from Field-Grown Sugar Maple Seedlings

The low survival of sugar maple (Acer saccharum) seedlings growing in unglaciated acidic forest soils (pH<4.0) in north-central Pennsylvania may be due in part to aluminum (Al) or manganese (Mn) phytotoxicity. Although foliar analysis has shown high concentrations (>2,000 ppm) of Mn, the location of potentially toxic elements in leaves or absorptive fine roots has not been studied in field-grown seedlings. This study of vascular (xylem and phloem) cells was conducted to determine by energy-dispersive X-ray microanalysis (EDX) whether Mn and/or Al could be detected in fine root or leaf midvein vascular cells. A secondary objective was to determine if vascular cells prepared by chemical fixation (CF) for conventional TEM or by freeze-substitution (FS) for EDX showed similar ultrastructural characteristics.Four sugar maple seedlings from an unglaciated field site (Potter County PA) were excavated, and roots were washed carefully. Two mm-long segments of fine roots and leaf midveins from each seedling were immersed in 3% glutaraldehyde for 6 hours, transferred to 0.1 M phosphate buffer, and transported to the Delaware Research Lab where tissues were post-fixed in OsO4 (in buffer), dehydrated in ethanol, and embedded in PolyBed-Araldite epoxy resin (CF method).

Development of peltate glandular trichomes of peppermint.

Cryofixation and conventional chemical fixation methods were employed to examine the ultrastructure of developing peltate glandular trichomes of peppermint (Mentha x piperita). Our results are discussed in relation to monoterpene production and the mechanism of essential oil secretion. Peltate glands arise as epidermal protuberances (initials) that divide asymmetrically to produce a vacuolate basal cell, a stalk cell, and a cytoplasmically dense apical cell. Further divisions of the apical cell produce a peltate trichome with one basal cell, one stalk cell, and eight glandular (secretory) disc cells. Presecretory gland cells resemble meristematic cells because they contain proplastids, small vacuoles, and large nuclei. The secretory phase coincides with the separation and filling of the sub-cuticular oil storage space, the maturation of glandular disc cell leucoplasts in which monoterpene biosynthesis is known to be initiated, and the formation of extensive smooth endoplasmic reticulum at which hydroxylation steps of the monoterpene biosynthetic pathway occur. The smooth endoplasmic reticulum of the secretory cells appears to form associations with both the leucoplasts and the plasma membrane bordering the sub-cuticular oil storage cavity, often contains densely staining material, and may be involved with the transport of the monoterpene-rich secretion product. Associated changes in the ultrastructure of the secretory stage stalk cell are also described, as is the ultrastructure of the fragile post-secretory gland for which cryofixation methods are particularly well suited for the preservation of organizational integrity.

Using High Pressure Freezing and Freeze Substitution to Investigate Kinetochore Ultrastructure in Vetebrate Somatic Cells

In vertebrate somatic cells, sister kinetochores attach chromosomes to the mitotic spindle by capturing microtubules (Mts) emanating from opposite spindle poles. Kinetochores are also required for chromosome alignment at the spindle equator, stabilizing kinetochore Mts, cell cycle control of anaphase onset, and poleward migration of sister chromatids during anaphase. Thus kinetochores play a critical role in the distribution of genetic information to daughter cells during cell division. Understanding the molecular mechanisms behind kinetochore function requires knowing how the molecular components are arranged relative to each other, and relative to the kinetochore Mts. Currently, however, our knowledge of kinetochore composition is incomplete, and until recently our knowledge of kinetochore ultrastructure was limited to the familiar trilamellar model derived from conventionally fixed and dehydrated specimens(Figures 1a&b).Highly fibrous structures, such as the kinetochore, are particularly vulnerable to distortions caused by the chemical fixation and dehydration methods used in conventional specimen preparations. Such distortions can be largely avoided by using high pressure freezing and freeze substitution (HPF/FS).

Overview of cell fixatives and cell membrane permeants.

The localization of proteins and carbohydrates within cells and tissues with specific antibodies has long been proven to be a valuable technique. Immunolocalization procedures allow one to detect not only well-characterized cellular structures but also provide information about newly characterized proteins and carbohydrates. This chapter will review some of the advantages and drawbacks of common chemical fixation and permeabilization methods used for immunolocalization at the level of light microscopy.

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.

Ultrastructure of the vitelline coat in the ascidians Phallusia mammillata , Ascidia mentula and Ciona intestinalis : new aspects revealed by freeze-substitution and deep-etching

The microstructure of the vitelline coat (VC) of the three ascidian species Phallusia mammillata, Ascidia mentula and Ciona intestinalis was analyzed by transmission electron microscopy (TEM) using freeze-substitution and deep-etching. In addition, eggs were examined by light microscopy and prepared by conventional chemical fixation methods for TEM in order to illustrate the advantages of cryopreparation for the preservation of the VC ultrastructure. In conventionally fixed eggs of P. mammillata and A. mentula the VC appeared as a tripartite structure, consisting of a thin outer fibrous layer (OFL), a central dense layer (CDL) and a relatively wide inner fibrous layer (IFL). In C. intestinalis a sparse OFL is made up by fine tufts of filaments but an IFL is lacking. In all three species the test cells were located in the area between the inner VC boundary and the egg cell membrane. Using our modified freeze-substitution method a clear improvement in preservation of the VC matrix was obtained. In all three species the OFL as well as the IFL in P. mammillata and A. mentula appeared as extended fibrous meshworks. Furthermore, the area of the perivitelline space, empty after conventional TEM preparation, was found to be filled by an additional matrix, designated here as perivitelline fibrous matrix (PVFM). Quick-freeze/deep-etch/rotary-shadowing enabled us to confirm the overall architecture of the egg matrix and allowed a further detailed investigation of the VC fine structure in P. mammillata. Accordingly, the vitelline coat of P. mammillata and A. mentula is a multilayered matrix composed of four distinct subunits (OFL, CDL, IFL, PVFM), whereas in C. intestinalis an IFL is lacking.

Sporicidal activity of chemical and physical tissue fixation methods.

AIMS: The effects of alcohol based fixation and microwave stimulated alcohol fixation were investigated on spores of Bacillus stearothermophilus and Bacillus subtilis (var. niger). METHODS: Spores were exposed to 10%...