Microwell Cluster for Processing Electron Microscope Sections for Immunocytochemistry

A microwell cluster was manufactured with silicone rubber for incubating thin sections for postembedding electron microscopic immunocytochemistry. The small size of the wells requires only minute amounts (as little as 5 μl) of antisera and other valuable immunoreagents. The capillary action of the wells holds the incubation media and grids in place, even if the cluster is turned upside down, thus facilitating safe transport and storage of the sections to be stained. This silicone rubber microwell cluster can also be used as a die to imprint wells in Parafilm sheets to be used for the same purpose.     

Quantitative Histochemistry; Design and Use of a Simple Microcell for Standardized Incubation on the Slide

Avoidance of stain variation among mounted sections incubated in small volumes of fluid can be overcome by the construction and use of a microcell. Perspex rings of appropriate diameter are attached to small Perspex rectangles to form wells, small trip bars being also attached. to the rectangles to simplify subsequent manipulation. The wells are filled with incubation fluid, and slides bearing frozen sections are held in position with small rubber bands. With certain dehydrogenases formazan production by this method is uniformly greater than that produced by incubation in drops of solution. In the case of the moderately active enzyme, 6-phosphogluconate dehydrogenase, the volume of the incubation mixture was not critical between 65 and 270 μl.     

Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum

We have developed a novel and simple method, requiring only a small amount of antigen, for removal of undesired antibodies from antiserum. The method was established using a well-characterized antiserum against rat luteinizing hormone (anti-rLH). Wells of polystyrene tissue culture plates were coated with rat LH (rLH). Anti-rLH diluted 1:3000 was added to rLH-coated wells and shaken to remove LH antibodies. Control anti-rLH was treated in a similar manner in non-rLH-coated wells. Both antisera were tested by immunocytochemistry on rat pituitaries. Antiserum from rLH-coated wells stained no cells, whereas the control serum stained cells that were morphologically typical of LH cells. The effectiveness of this antibody removal was also confirmed in a modified ELISA. In another experiment, anti-rLH and anti-hTSH beta sera were mixed. The final dilution of both antisera was 1:10,000. Anti-rLH was removed by the purification method described. Completeness of antibody removal was confirmed by a double-immunohistochemical staining of rat pituitary in which sections were first stained by the PAP method and then stained with an immunofluorescence procedure after elution of the first antigen-antibody complex. The mixed antiserum incubated in rLH-coated wells did not stain LH cells. There was no co-localization between the LH immunopositivity demonstrated by an anti-rLH serum using immunofluorescence and cells immunostained with the purified antiserum using the PAP method. As indicated in ELISA, the titer of the TSH beta antiserum was not decreased compared to that of the untreated, mixed control antiserum, and the LH antibodies were eliminated by the treatment. This new purification method has four distinct advantages: (a) antiserum is not treated chemically; (b) it requires only a small amount of antigen compared with the amount required for affinity chromatography; (c) neither the undesired antigen-antibody complex(es) nor an excess amount of antigen is present in the purified antiserum; and (d) removal of undesired antibodies can be monitored by ELISA.     

Development of a miniaturized four-culture method for the rapid enumeration of four bacterial groups in ground beef

AIMS: A simplified and rapid method was developed to identify and enumerate total mesophilic microbial load, gram-negative bacteria, coliforms, and Escherichia coli in ground beef, using a single module. METHODS AND RESULTS: A 96-well microtiter plate was divided into four sections of two lanes (24 wells), with each section dedicated to each of the four critical bacterial groups. Double strength of four different selective broths (100 microl) was added to each well and an aliquot of sample (100 microl) from the first well was transferred to the next to achieve a twofold serial dilution in each series (24 wells). Initial bacterial load was calculated using the final consecutive positive reaction exhibiting color change or fluorescence. To validate the method, ground beef (n = 32), inoculated with a three-strain mixture of E. coli, together with 30 samples of commercial ground beef, were assayed by the four-culture method and conventional plating. For all media tested, the coefficients of determinations (r2) between the results derived from the four-culture method and those of conventional plating method ranged from 0.83 to 0.92. CONCLUSIONS: The high degree of association between the two methods indicates that the four-culture method could be easily applied to the enumeration of four critical bacterial groups in ground beef. SIGNIFICANCE AND IMPACT OF THE STUDY: This method will facilitate sanitation monitoring at meat processing plants by shortening time required for results, reducing consumable material costs, simplifying procedures, reducing the space required, and cutting the cost of start up equipment.     

The quantitative assay of the clustering activity of the lymphocytosis-promoting factor (pertussis toxin) of Bordetella pertussis on Chinese hamster ovary (CHO) cells

An experimental design and a statistical method for the estimation of the clustering-response activity of lymphocytosis-promoting factor (LPF) in Chinese hamster ovary cells growing in wells on a microplate were investigated. The scoring method introduced by Ipsen was adopted to express the grade of the clustering response rather than the end-point method generally used. The scoring method was validated by statistical analyses. The grade of response varied with the location of the wells on a microplate, and thus the expression of the clustering activity of a test sample in terms of the end-point may be inadequate in terms of accuracy and reproducibility. It was shown that the allocation of test samples to individual wells according to a Latin square design minimized the effect of the location of wells on the clustering response. Under such experimental conditions, a fairly precise and reproducible method for the quantification of the clustering activity was developed.     

Influence of 2,3,5-Triiodobenzoic Acid and 1-N-Naphthylphthalamic Acid on Indoleacetic Acid Transport in Carnation Cuttings: Relationship with Rooting

³H-IAA transport in excised sections of carnation cuttings was studied by using two receiver systems for recovery of transported radioactivity: agar blocks (A) and wells containing a buffer solution (B). When receivers were periodically renewed, transport continued for up to 8 h and ceased before 24 h. If receivers were not renewed, IAA transport decreased drastically due to immobilization in the base of the sections. TIBA was as effective as NPA in inhibiting the basipetal transport irrespective of the application site (the basal or the apical side of sections). The polarity of IAA transport was determined by measuring the polar ratio (basipetal/acropetal) and the inhibition caused by TIBA or NPA. The polar ratio varied with receiver, whereas the inhibition by TIBA or NPA was similar. Distribution of immobilized radioactivity along the sections after a transport period of 24 h showed that the application of TIBA to the apical side or NPA to the basal side of sections, increased the radioactivity in zones further from the application site, which agrees with a basipetal and acropetal movement of TIBA and NPA, respectively. The existence of a slow acropetal movement of the inhibitor was confirmed by using ³H-NPA. From the results obtained, a methodological approach is proposed to measure the variations in polar auxin transport. This method was used to investigate whether the variations in rooting observed during the cold storage of cuttings might be related to changes in polar auxin transport. As the storage period increased, a decrease in intensity and polarity of auxin transport occurred, which was accompanied by a delay in the formation and growth of adventitious roots, confirming the involvement of polar auxin transport in supplying the auxin for rooting. Received April 19, 1999; accepted December 2, 1999     

A novel immunohistochemical technique for the detection of human oral carcinomas transplanted in nude mice using mouse monoclonal antibodies

 We have developed a new immunostaining technique specifically for the detection of human tumors transplanted into nude mice using mouse monoclonal antibodies (MoAbs) produced in our laboratory. The formation of a molecular complex consisting of three components (mouse MoAb or hybridoma supernatant, biotin-labeled anti-mouse immunoglobulins, and normal nude mouse serum) markedly reduced background staining and enhanced specific reaction with the transplanted tumors in nude mice. When MoAb production by electrofusion was screened with this new method, the incidence of hybridoma supernatant reactive with sections of transplanted tumor was 2.3 per 100 wells immunostained. These results suggest that production of MoAbs using transplanted tumors is immunohistochemically possible and that this method may provide a new means for developing useful tumor markers. Accepted: 30 September 1996     

Improvement of the Teflonized Slides Used in the Immunofluoresgent Antibody Technique

The indirect fluorescent antibody (IF) test is often performed on glass slides prepared with smears or tissue sections containing an antigen. It is convenient to have several separate and distinct areas of antigen on the same slide, e.g. to compare dilution steps, and to save time when producing the antigen slides. With this end in view a technique for preparing slides with wells in a thin film of teflon-like compound has been elaborated (Goldman 1968). This technique is useful when a great number of slides need to be prepared in advance, e.g. to test a substantial amount of material. It has, however, not been widely used im serology for Babesia, perhaps because any teflon remaining within the wells is stained with the fluorescent dye, thus creating a potentially disturbing background.     

Extrapolation of depositional geometries of the Menorcan Miocene carbonate ramp with ground-penetrating radar

The Tortonian carbonate ramp of Menorca was previously studied on the basis of outcrops along sea-cliff outcrops. These sea cliffs, in combination with inland water wells, are the basis for a facies model for the reconstruction of the internal architecture and for characterizing the internal heterogeneities of this carbonate platform. However, any such three-dimensional reconstruction is generally limited by the given geometrical arrangement of the two-dimensional outcrops and the uncertainties of correlation with the one-dimensional wells. Here, ground-penetrating radar (GPR) has been employed in order to test and refine the depositional model. Although GPR is well known for being an excellent tool for high-resolution underground studies of sedimentary systems, the application for studying carbonate rocks is still far from routine. The reason for this discrepancy is two-fold: the minor mineralogical contrast between lithologies in carbonate rocks results in subtle reflections, and, even more important, the porosity structure in carbonates is thoroughly and repeatedly changed during diagenesis, commonly across the different facies, leading to problems in predictability of the petrophysical properties. The study of the Menorcan carbonate ramp with large distance–deep penetration GPR sections demonstrates that in spite of these difficulties, GPR is a valuable tool for extrapolating information from outcrops and wells. It is useful for characterizing heterogeneities larger than outcrop scale.     

Les associations des calpionelles du Berriasien et du Valanginien inférieur de la Tunisie septentrionale. Comparaison avec les associations du Rif externe (Maroc)

The study of ten thin sections from outcrops from Lower Cretaceous deposits and several thin sections obtained from three petroleum wells, allow us to better characterize the associations of calpionellid zones and subzones of the Berriasian and Lower Valanginian in northern Tunisia. The B and C zones, respectively, show three and two levels with calpionellid associations corresponding to B1, B2, B3 and C1, C2 subzones. In the D zone, associations of the D1 and D2 subzones were identified. The E zone has been characterized for the first time in Tunisia and in southern-Tethyan margin of North Africa, on the basis of its index specie and associated species.     

A new method of sample application for horizontal slab polyacrylamide gel electrophoresis

A new method of sample application for horizontal slab polyacrylamide gel electrophoresis has been developed which solves the main problems associated with existing systems. A quick, simple procedure is described for placing a dry powder mixture of Celite and Sephadex into sample wells of any shape cut to the full depth of the gel slab. Samples can then be added to the powder to form a moist firm bed of material in the wells which prevents leakage of sample from the well. The method enables the quantitative electrophoresis of many samples with widely differing concentrations and volumes without the problems of electrodecantation, loss of electrical contact through the wells, or uneven penetration of sample through the full thickness of the gel.     

A method for the preparation of serial thick sections of plastic-embedded plant tissues.

A procedure is described in which thick sections (2-10 mu or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, alkalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pre-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Estimating elephant densities from wells and droppings in dried out riverbeds

In this paper, we present a new method for estimating elephant densities by counting elephant wells and dung boli within dry seasonal flooding rivers. A combination of aerial and ground counts of elephant wells and dung boli in the Ewaso Ngiro River were related to elephant numbers, obtained from an on‐going monitoring program of individually identified elephants in Samburu and Buffalo Spring National Reserves, Kenya. The total number of elephant observations was highly correlated with both densities of wells and dung boli at a spatial resolution of 4‐km river‐section. This indicates that both wells and droppings can be used for estimating relative densities at such spatial resolution. The method can be used as a quick and reliable way of estimating relative elephant densities in semiarid regions but is sensitive to differences in the time when different parts of the river dry out and will be unreliable in areas with secondary un‐censused water sources. A short 4‐week period between the river dry out and the count is recommended, because of an error induced by a level of well reuse and the difficulties in counting areas of high well densities from the air.     

Enzyme-Linked Immunosorbent Assay of Fungal NADP-Glutamate Dehydrogenase

A sensitive and reliable method has been developed for the quantitation of NADP⁺-glutamate dehydrogenase from the phytopathogenic Ascomycete Sphaerostilbe repens using a two-step competitive enzyme-linked immunosorbent assay. Purified enzyme was adsorbed noncovalently to polystyrene wells and rabbit immunserum was allowed to bind to antigensensitized wells. Bound specific antibody was visualized by goat antirabbit immunoglobulin covalently linked to alkaline phosphatase using paranitrophenylphosphate as the substrate. Increasing amounts of purified enzyme or crude fungal extracts were quantitated by their ability to inhibit specific antibody adsorption to antigen-coated polystyrene wells. This system proves to be useful in the range of 10 to 80 nanograms of enzyme level. Using this assay, identical amounts of NADP⁺-glutamate dehydrogenase were found in mycelia grown on nitrate and ammonia sources.     

Safranin O Staining Using a Microwave Oven

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.     

Safranin O Staining Using a Microwave Oven

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.     

Tridimensional structure of the Golgi apparatus in type A ganglion cells of the rat

The three-dimensional structure of the whole Golgi apparatus and of its components in type A ganglion cells was examined in thin and thick sections by low- and high-voltage electron microscopy. At low magnification, in 10-micron-thick sections of osmicated cells, the Golgi apparatus formed a broad, continuous perinuclear network. At higher magnification and in thinner sections of cells impregnated with uranyl acetate-lead-copper citrate or postfixed in K-ferrocyanide-reduced osmium, the Golgi apparatus appeared as a heterogeneous structure in which saccular regions characterized by stacks of saccules alternated with intersaccular regions made up of branching membranous tubules which bridged the saccules of adjacent stacks. The saccular regions consisted of the following superimposed elements: a cis-osmiophilic element made up of anastomosing tubules; two or three saccules negative for the phosphatases tested (i.e., nicotinamide adenine dinucleotide phosphatase = NADPase, thiamine pyrophosphatase = TPPase, and cytidine monophosphatase = CMPase); two saccules showing TPPase activity; and one to three trans-sacculotubular elements showing a "peeling-off" configuration, one of which showed CMPase activity. The saccules (phosphatase-negative) on the cis-side of the Golgi stacks showed, in addition to small circular pores, larger perforations in register. The cavities thus formed in the stacks of saccules, called "wells," always associated with small 80-nm vesicles, had a pan shape with the mouth directed toward the cis-face and the bottom closed by a TPPase-positive saccule. In face views of the saccules, the smallest of these perforations showed either a crescent shape, due to the presence of a bud on one side of the perforation, or a circular shape with a single small 80-nm vesicle in the center which was occasionally attached to the saccule by a filiform stalk. Such smaller cavities were considered as the precursors of the larger perforations and eventually of the wells. The small 80-nm vesicles seen in the small cavities or in the wells appeared to form in situ and possibly migrate toward the cisternae of endoplasmic reticulum seen proximal to the cis-face of the stack of saccules. Small 80-nm vesicles were also numerous in the intersaccular regions, along the lateral- and trans-aspects of the Golgi stacks, while larger, 150-to 300-nm vesicles, coated and uncoated, were seen only on the trans-face of the Golgi stacks in proximity to the trans-sacculotubular elements which appear to "peel off" from the Golgi stacks.     

Specific identification of free and esterified fatty acids in tissue sections

Synopsis The histochemical method of Adamset al. (1966) for demonstrating triglycerides in tissue sections was applied to kidneys exhibiting a wide variety of disease states. It became apparent, as would be expected, that the existing method demonstrates not only triglycerides but also free fatty acids in the same section. Even though the presence of free fatty acids could be detected in the control sections, their existence made it impossible to identify triglycerides with certainty.A modification is described which employs a potassium hydroxide-dioxan mixture to saponify and extract selectively free fatty acids from tissue sections. Fatty acids in free form can be demonstrated separately, in parallel sections, from those esterified as triglyceride. This modified technique was applied to frozen sections of formalin-fixed human and rat tissues, revealing distinct and highly characteristic distribution patterns for these two forms of fatty acid.     

Immunohistochemical procedures for the demonstration of peptide- and tyrosine hydroxylase-containing nerve fibers in cryostat sections of unfixed rapidly frozen tissue stored for long periods of time. A study on heart tissue

Traditional protocols for the immunohistochemical localization of peptides and tyrosine hydroxylase (TH) in nerve fibers in cryostat sections require the tissue to be thoroughly fixed and rinsed and to be processed for the cryostat sectioning and the immunohistochemical staining more or less directly after freezing. In the present study it was tested whether also unfixed, rapidly frozen tissue, conforming to guinea pig and bovine heart specimens, can be used for the visualization of neuropeptides [neuropeptide Y (NPY) and substance P (S P)] and TH in cryostat sections. The following observations were made: 1) NPY-immunoreactive (IR) and S P-IR nerve fibers could be clearly identified in both fixed and unfixed sections of this type of tissue. 2) TH-IR nerve fibers could be detected in unfixed tissue if the sections were post-fixed with aldehydes by the use of a two-step fixation process related to a sudden change of pH. However, the outlines of the nerve fibers were sometimes diffuse. 3) Storage of unfixed tissue for periods of up to 2.5 years at-80 degrees C did not lead to a decrease in immunoreactivity. 4) Somewhat higher concentrations of primary antibodies had to be used for sections of unfixed tissue than for sections of fixed tissue when the FITC method was used. This waste of antibodies was partly overcome by use of the biotin-streptavidin method. The glyoxylic acid induced catecholamine(CA)-fluorescence method for demonstration of sympathetic nerve fibers was also applied and was found to give optimal results after storage of tissue for up to 2.5 years.(ABSTRACT TRUNCATED AT 250 WORDS)