Slide Mounts Research Articles

Agar-Gelatin for Embedding Tissues Prior to Paraffin Processing

Tissue Pre-Embedding Histologists often arrange tissue samples in a very specific orientation prior to paraffin embedding using a process known as pre-embedding. Pre-embedding has traditionally utilized molten agar that is poured over the tissue and which hardens to maintain the proper orientation of the tissue during subsequent embedding procedures. A shortcoming of using agar as the pre-embedding media is that certain tissues shrink during the embedding process, and the agar-based pre-embedding media limits tissue expansion during slide mounting, resulting in difficulties with the tissue sample adhering to the microscope slide. Jones and Calabresi have provided a simple solution to this challenge by using agar mixed with gelatin as a new pre-embedding media. The authors demonstrate that for central nervous system tissues, which tend to shrink during embedding, using agar-gelatin pre-embedding allows these tissues to completely expand when placed on a 42°C water bath prior to slide mounting. The gelatin ...

Combining the gold chloride and toluidine blue stains to investigate chick embryo neural tissue

Toluidine blue and gold chloride stains are both well-known for staining nervous tissue. Toluidine blue is a general method to stain neurons and glia, used because of the speed of action. Gold sublimate is a delicate procedure that requires extreme purity of reagents and materials, and it stains astrocytes and neurons. The aim of this study was to combine these two special staining methods for embryonic neural tissue and to obtain a repeatable and easily manageable new staining method with improved overall visualization of neural tissue. Fertilized Broiler hatching eggs were incubated at 36 degrees C and on day 8 prepared for histology. Sections were cut at 7 microm thickness and slides were stained for 30 s using a 0.1% Toluidine blue solution. These slides were rinsed with dH2O followed by placing them in gold sublimate for 4 h at room temperature in the dark. Rinsing with dH2O and transferal to a 5% sodium thiosulfate solution was done, followed by another rinsing and mounting of slides. Results showed that the combination of the two methods offers a fast, reliable method with which chick embryonic neural tissue can be evaluated.

Nitrocellulose as a General Tool for Fungal Slide Mounts

The microscopic examination of fungi is conventionally carried out by wet mount staining using lactophenol cotton blue, in combination with different modes of sample preparation: the use of mounting needles, the “adhesive tape lift,” and various agar block techniques are well documented in the

Laser capture microdissection of plant cells from tape‐transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling

Laser capture microdissection and related technologies permit the harvest of individual cells and cell types. Isolation of either nucleic acids or proteins from laser-captured cells supports such downstream applications as the construction of cell-specific cDNA libraries and the profiling of expressed genes and proteins. The success of these endeavors is dependent upon the yield, purity and structural integrity of the macromolecules derived from harvested cells. Here, we report protocols that promote the isolation of structurally intact RNA from laser-captured cells of paraffin-embedded tissues. The use of a tape transfer system that obviates the need to wet paraffin sections prior to slide mounting significantly increases RNA structural quality. Integrity is assessed directly via electrophoretic separation of picogram-nanogram levels of total RNA isolated from multiple cell types, including those comprising Arabidopsis ovules, replums and stamen abscission zones. RNA prepared from specialized cells within siliques provided targets for profiling the Arabidopsis genome during replum cell development. Digital northern analysis of transcripts expressed near the threshold of the system's ability to score signal presence suggests that low-abundance transcripts representing as little as approximately 0.002% of total mRNA can be reliably detected. Microarray data reveal a significant shift from primary cell-wall metabolism to lignin biosynthesis in replum tissues during fruit maturation.

A New Method for Preparing Slide Mounts of Whole Bodies of Microlepidoptera

A new method is described for preparing slide mounts of whole bodies of microlepidoptera to facilitate comparative morphological studies. This method conserves traditional characters of wing pattern while revealing wing venation and other morphological structures of the denuded body. Examples of new characters revealed on slide mounts of whole bodies and photographed with a Confocal Laser Scanning Microscope are given for selected species of Gelechioidea. Also, the historical use of morphological characters for defining taxa of Lepidoptera is briefly reviewed.

Biological Monitoring of Solar UV Radiation at 17 Sites in Asia, Europe and South America from 1999 to 2004

A small and robust dosimeter for determining the biologically effective dose of ambient UV radiation has been developed using UV-sensitive mutant spores of Bacillus subtilis strain TKJ6312. A membrane filter with four spots of the spores was snapped to a slide mount. The slide was wrapped and covered with two or more layers of polyethylene sheet to protect the sample from rain and snow and to reduce monthly-cumulative doses within the measurable range. From 1999, monthly data were collected at 17 sites for more than 1 year, and data for 4 to 6 consecutive years were obtained from 12 sites. Yearly total values of the spore inactivation dose (SID) ranged from 3200 at subarctic Oulu to 96 000 at tropical Denpasar, and the mean yearly values of SID exhibited an exponential dependence on latitude in both hemispheres with a doubling for about every 14 degrees of change. During the observation period, increasing trends of UV doses have been observed at all sites with more than 5 years of data available. Year-to-year variations at high and middle latitude sites are considered due mostly to climatic variation. At three tropical sites, negative correlations between the yearly doses and the column ozone amounts were observed. The results verified the applicability of spore dosimetry for global and long-time monitoring of solar UV radiation, in particular at tropical sites where no monitoring is taking place.

Basic Fibroblast Growth Factor (bFGF, FGF-2) Potentiates Leukocyte Recruitment to Inflammation by Enhancing Endothelial Adhesion Molecule Expression

Basic fibroblast growth factor (bFGF, FGF-2) is a potent angiogenic factor and endothelial cell mitogen. Although bFGF levels are increased in chronically inflamed tissue, its role in inflammation is unclear. We investigated the effect of bFGF on acute dermal inflammation and the recruitment of monocytes, T cells, and neutrophils. Leukocyte recruitment to inflamed sites was quantified with radiolabeled leukocytes. Intradermal injection of bFGF in rats did not induce leukocyte recruitment or inflammation. However, the recruitment of leukocytes to inflammation induced by tumor necrosis factor-alpha, interferon-gamma, C5a, or a delayed hypersensitivity reaction was enhanced by bFGF by 55 to 132% (P < 0.05). Either acute or prolonged bFGF treatment of dermal sites had this effect. The potentiating effect of bFGF on leukocyte recruitment was also seen in joints. There was no associated modulation of vascular permeability, blood flow, or angiogenesis in the sites by bFGF. However, the expression of the endothelial cell adhesion molecules (CAMs) for leukocytes, P-selectin, E-selectin, and ICAM-1, was significantly up-regulated in the inflamed tissue by bFGF, as quantified by radiolabeled anti-CAM antibody binding in vivo. Thus, although not directly proinflammatory, bFGF synergistically potentiates inflammatory mediator-induced leukocyte recruitment, at least in part, by enhancing CAM up-regulation on endothelium.

Evaluating the eye's rotational stability during standard photography

To evaluate the rotational stability of the eye during standard photography and determine its effect on the measurement of toric intraocular lens (IOL) orientation. Department of Ophthalmology, University Erlangen-Nuremberg, Erlangen, Germany. The rotational stability of the eye was evaluated using standard photographs taken with a telecentric fundus camera (Zeiss). Two sets of fundus images were taken at least 6 months apart in 400 eyes of 200 patients. The axial position of the eye was determined using 2 characteristic markers of the fundus. The angle between the 2 images (autorotation angle) was measured in each eye. The mean absolute autorotation was 2.3 degrees +/- 1.7 (SD) (range 0 to 11.5 degrees). Nine percent of eyes did not rotate. The rotation was less than 3 degrees in 55% of eyes and was 3 degrees or greater in 36% of eyes. Eyes of patients younger than 50 years rotated less than eyes in older patients (mean 2.2 +/- 1.5 degrees and 2.5 +/- 1.8 degrees, respectively) (P=.04). A visual acuity of 20/20 or better (P=.02) and a refractive cylinder of less than 1.75 diopters (P=.01) were correlated with smaller amounts of autorotation. Potential causes of artificial eye rotation induced by the photographic technique included camera adaptation (3-degree intrinsic error), slide mounting (<1 degree), slide projection (<0.5 degree), marking of characteristic fundus details (<1 degree), and head inclination. Cyclorotation of the eye during standard photography may lead to overestimation or underestimation of the presumed spontaneous rotation of an implanted toric IOL. Results show that 11.5 degrees of toric IOL rotation would lead to residual astigmatism that is 40% of the initial astigmatic power and 3 degrees, 10% of the initial power. Digital imaging may reduce the intrinsic errors of standard photography.

Prevalencia de microsporidios y otros parásitos intestinales en pacientes con infección por VIH, Bogotá, 2001.

Opportunistic intestinal parasites are a common cause of diarrhea in HIV-infected patients. To determine the prevalence of microsporidia and other opportunistic parasites infecting HIV patients in Bogotá, Colombia, 115 patients were examined for these infections during the year 2001. The institution and the sample percent from each are as follows: Santa Clara Hospital, 33.0%; San Pedro Claver, 20.0%; Simón Bolívar Hospital, 14.8%; San José Hospital, 13.9%; Central de la Policía Hospital, 6.1%; Compensar, 5.2%; Colombian League against AIDS, 2.6%; San Ignacio Hospital, 2.6%, and the Military Hospital, 1.7%. The average patient age was 36 years, with a range from 18 to 71 years. Patients with complaint of gastrointestinal symptoms were asked to provide two consecutive stool samples. The samples were concentrated in formalin-ether and examined microscopically for intestinal coccidian parasites by direct wet slide mounts. The prevalence of intestinal opportunistic parasites was 10.4% for Cryptosporidium sp. Initially, 29% of the samples were found to be positive for microsporidian spores using a modified Ziehl Neelsen chromotrope stain, but only 3.5% of them were confirmed as positive when a calcofluor/Gram chromotrope stain was used. The general prevalence of intestinal parasites was 59.1%. The most frequently found pathogens were Blastocystis hominis, 25.2%, and Entamoeba histolytica, 13%. In other studies with HIV patients in Colombia, lower prevalences of Cryptosporidium sp. infection were observed.

123 Permanent Fluorescent Nuclear Staining of Binucleate Dinoflagellates

Typically, fluorescent microscopy of dinoflagellate nuclei is of poor resolution, due mainly to visual obstruction of the nuclei by plastids, pigment granules, and thecal plates. Moreover, the usual slide mounts using buffered glycerol are temporary, and fade after a week or so. We have developed a procedure to clear pigments from dinoflagellates, followed by fluorescent staining of the nuclei. The cells are then prepared as permanent mounts using an ultraviolet light‐catalyzed resin to produce stained samples which may be kept for at least three years with little loss of fluorescence. This procedure can also be used to prepare plastic embedded dinoflagellate cells which can then be sectioned at 1–2 nm, fluorescent stained, and permanently mounted. Suitable nuclear stains are DAPI, Hoechst 33258, ethidium bromide and acridine orange. The dinoflagellate (dinokaryotic), and endosymbiont (eukaryotic) nuclei are clearly visualized, revealing individual chromosomes in the dinoflagellate nucleus, and a highly lobed morphology of the endosymbiont nucleus.

Patency File and Apical Transportation: An In Vitro Study

The purpose of this study was to evaluate the transportation produced at the apical foramen by the use of stainless steel and nickel-titanium K-files #10, #15, #20, and #25 as a patency file. Thirty human maxillary lateral incisors were transversally cut at 4 mm from the apex and mounted in square pieces of silicone with special marks to ensure repositioning of the specimens. The specimens were randomly divided in two equal groups of 15. In group A, stainless steel K-files #10, #15, #20, and #25 were used as a patency file. In group B, a stainless steel K-file #10 followed by nickel-titanium K-files #15, #20, and #25 were used as in group A. Sodium hypochlorite was used as the irrigating solution. Photographic slides of the apical foramen of each specimen before instrumentation and after the use of each file size were taken. The photographic slides of each specimen were mounted in slide mounts, projected onto the same drawing paper, and superimposed according to the peripheral shape of the root. Transportation of the apical foramen was determined comparing the five drawings of each specimen. Transportation was detected in 18 of the 30 specimens; 9 in group A and 9 in group B. No statistically significant differences were seen between groups (p = 1.00).

Characterization of light penetration in rat tissues.

The goal of this study is to determine the optical properties of different rat tissues with respect to spatial intensity variation and light distribution. We are interested mainly in the wavelength of 630 nm. Nevertheless, for liver tissue we have used 514 nm and 670 nm as well. In the past, many articles have been written about the interaction of lasers with rat tissues. However, the technique of imaging the light distribution allows us to obtain the spatial scattering as well as an effective attenuation coefficient for the light intensity. Slices of different rat tissues were placed between two microscope slide mounts (spaced by 3 mm). A laser beam was irradiated on the sandwiched tissue. A CCD camera placed on the side, orthogonal to the beam path, recorded the intensity distribution of the scattered light. Analysis of this spatial intensity profile allowed determining the variation of the intensity as the light penetrates the tissue. We have found that abdominal wall fat presents the lowest exponential decay when compared with liver, muscle, and kidney. The obtained values provided good data about the light distribution in those tissues when irradiated with a nondiffuse laser beam. For all tissues, we observed a spherical light distribution and exponential decay. Cirrhotic liver shows much stronger decay than healthy liver. These results are useful for several applications of laser for biostimulation a phototherapy.

Book Review

Book Review

Chapter 13 Nuclear and Cortical Histology for Brightfield Microscopy

A number of “classic” ciliate stains have been developed, handed down, and improved in ways that have enabled researchers to visualize cortical morphology and nuclear configurations in permanent slide mounts of ciliated protozoans using conventional brightfield microscopy. This chapter describes three of these procedures because they are commonly used with the ciliate Tetrahymena thermophila . The Chatton–Lwoff silver stain for Tetrahymena is used to visualize patterns of basal bodies within the ciliary rows and oral apparatus as well as contractile vacuoles in a wide variety of ciliates. Protargol staining produces exquisite permanent samples of cortical microtubule staining along with nuclear staining. This procedure requires practice and “fiddling” to achieve the highest quality preparations. Giemsa stain is a reliable method for producing permanent slides revealing nuclear configurations in growing or conjugating cells.

Intravascular stents: a new technique for tissue processing for histology, immunohistochemistry, and transmission electron microscopy

BackgroundStudy of the vascular response to stent implantation has been hampered by difficulties in sectioning metal and tissue without distortion of the tissue stent interface. The metal is often removed...

Creating low-power photomicrographs using a 35 mm digital slide scanner.

A method for obtaining high quality photomicrographs at very low power magnification using a digital slide scanner is described. Using an easily modified 35-mm film slide mount, it is possible to scan a conventional 25-mm wide glass slide and to obtain an image that is uniformly in focus and of even illumination. The computer bitmap file that is generated is suitable for computer presentations or publication-quality prints and may be magnified up to 12-fold without significant image degradation.

Ultrastructure and geochemistry of endolithic microorganisms in limestone of the Niagara Escarpment

Endolithic microbial communities dominated by photosynthetic cyanobacteria occur at a depth of 0.5 – 3.0 mm inside dolomitic limestone that forms the vertical cliffs of the Niagara Escarpment in Ontario, Canada. Coccoid Gloeocapsa was the most prominent cyanobacterial species in colonized rock samples examined by differential interference contrast and episcopic fluorescent microscopy. Filamentous varieties of cyanobacteria were less common than coccoid forms but could be easily distinguished in all slide mount preparations. Scanning electron microscopy showed that the endoliths occurred in communal populations situated within the open pore spaces of the limestone. Heterotrophic bacteria were also observed growing as epiphytes on dead and living cyanobacteria, and in epilithic biofilms on pore space walls. In thin-sectioned specimens examined by transmission electron microscopy, these adherent bacteria commonly exhibited extensive accumulations of fibrous extracellular polymeric materials external to their cell walls. Biomass concentrations, estimated from the organic phosphorus content of the samples, generally paralleled rock porosity and ranged from 3.3 to 17.1% dry weight. Carbon isotope (14C) measurements indicated that atmospheric carbon dioxide is used by the endolithic cyanobacteria, rather than dissolved inorganic carbon from the weathering of carbonate minerals in the limestone host rock. In addition, whole rock multielement analyses revealed an enrichment of some elements (e.g., phosphorus, barium, lead, and zinc) in the endolith zone over the host rock, while other elements (e.g., magnesium, calcium, iron, and copper) were depleted. The implication is that the endolithic microorganisms play an active role in the biogeochemical cycling of nutrient and trace elements in cliff face ecosystems of the Niagara Escarpment.Key words: limestone, endolith, biofilm, cyanobacteria, geochemistry.

Melanin content and distribution in the surface corneocyte with skin phototypes

An individual's sensitivity to sunlight is traditionally assessed by the Boston or Fitzpatrick classification of skin type. The ability to tan depends, to some degree, on the melanin content of the epidermis. In the study reported here, surface corneocytes in exposed skin and unexposed skin have been assessed using a surface stripping slide mounting technique and an Optomax V image analyser, with which the percentage of corneocyte area occupied by melanin granules has been taken as the melanin content index (MCI). There was a significantly different MCI between different skin types for both exposed (P < 0.0001) and unexposed (P < 0.0001) areas using the Kruskal-Wallis one-way ANOVA test. There was also a positive significant correlation between MCI and skin types II-VI in both exposed (r = 0.95, P < 0.001) and unexposed areas (r = 0.89, P < 0.005). Image analysis also demonstrated that the number of melanin granules in surface corneocytes was significantly higher in the exposed area compared with the unexposed area, for skin types II, III, IV, V and VI. Melanin cap-like structures were also observed in exposed corneocytes and heavily pigmented skin contained larger melanin particles than fairer skin. The results indicate that an individual's skin phototype and melanin content, assessed by image analysis, have a significant correlation.

The use of selective enrichment for the isolation of species-specific DNA probes for insects.

AbstractThe identification of insects and other invertebrates is often extremely difficult if not impossible to achieve when attempted by traditional methods. There are several reasons why these difficulties are encountered. They may be owing to the presence of species complexes where individuals of several closely related species are morphologically indistinguishable. The species may be too small to identify easily without slide mounting or internal examination, thereby making identification extremely time consuming and labor intensive, or it may be that immature stages including eggs need to be identified. In this latter instance very few identification keys exist for juveniles and therefore often the only course is to attempt to rear the juvenile to the adult stage. Occasions also may arise where large numbers of individuals need to be screened, and, should difficulty in identification be experienced, the work may be rendered impracticable.KeywordsPolymerase Chain ReactionSterile Distil WaterAdapter MoleculeSelective EnrichmentMolecular Biology GradeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Improvement of a 500 keV heavy-ion-beam probe for JIPP T-IIU tokamak

Several improvements in the high-voltage heavy-ion-beam probe (HIBP) are discussed. (1) It is clearly found that the precision slide mount of the detector plates 30° parallel to the base electrode is very effective for the determination of the in-plane entrance angle of the beam in the analyzer to estimate the error in the potential measurement. (2) A two-staged optical trap in the HIBP greatly reduced the effect of the UV radiation in the analyzer. (3) A multiple-plate detector up to 13 measurement points clearly showed the direction of the propagation of the turbulence and path-integral effects.