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1.
Tissue preservation, and immunogold cytochemical and in-situ hybridization labelling intensities vary according to the preparatory protocols used. We wished to determine which preparative protocols produce optimal preservation, protein and mRNA labelling. Nine combinations of fixative and embedding resin were therefore studied using postembedding immunoelectron microscopy and a novel immunogold digoxygenin in situ hybridization (ISH) system, to quantitate the presence of transforming growth factor beta 1 (TGF 1) protein and message in human skin. The best preservation was observed in tissue fixed in 1% glutaraldehyde and embedded in LR White resin or low acid glycolmethacrylate resin (LA-GMA). Preservation was poor in tissue fixed with 1% glutaraldehyde and fair in 4% paraformaldeyde, when embedded in Unicryl. Ethanediol dehydration coupled with LA-GMA embedding resulted in reasonable preservation. Based on quantitative measures of the labelling density for TGF 1 protein and mRNA, immunogold labelling was adequate with 1% glutaraldehyde fixation coupled with LR White or LA-GMA resins, and also with 4% paraformaldehyde and LR White resin, but was best with ethanediol dehydration and LA-GMA embedding. ISH labelling under basal conditions was best in LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde. The ISH label in tissue fixed with 1% glutaraldehyde and embedded in LA-GMA was significantly increased by treatment with proteinase K. Overall, ethanediol dehydration was associated with a good immunoelectron microscopic (IEM) label while LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde resulted in a consistently detectable ISH label. LA-GMA embedding with 1% glutaraldehyde fixation gave a good result with both IEM and ISH labelling.  相似文献   

2.
Summary A method is described for the histochemical demonstration of -glutamyl transpeptidase in tissue sections embedded in glycol methacrylate at low temperature. Enzyme activity was preserved by a short (3 h) fixation of tissue in 4% paraformaldehyde at 4° C prior to embedding at 4° C. Tissue embedded in glycol methacrylate combined good morphology with accurate enzyme localization. Blocks of the embedded tissue could be stored at room temperature for at least 3 months without loss of enzyme activity. The resin is non-fluorescent, allowing the use of the fluorescent coupling agent 5-nitrosalicylaldehyde to visualize the reaction product.  相似文献   

3.
Precise sampling from whole lobes of mouse lungs fixed in the inflated state and embedded in epoxy resin can be not only feasible but also efficient. A 1 μm section is cut from an embedded lobe with a rotary microtome and a steel knife. This section is stained and photographed, and from it a 35 × enlarged print is prepared. A grid of transparent plastic scored with 35 mm squares, lettered vertically and numbered horizontally, is superimposed over the photograph. The area chosen for electron microscopy thus becomes identifiable by a letter-number designation obtained from the grid. This area is then located by light microscopy on a 2 mm slice taken from the block from which the 1 μm section was cut, by use of oblique illumination and the calibrated mechanical stage of the light microscope. A block of 1.3 mm diameter is removed for electron microscopy from the tissue by a rotatable circular spring-loaded punch screwed into the objective turret of the microscope. The removed cylinder is mounted on a metal stub and ultrathin sections cut from the faced tissue. The method is as equally suitable for the examination of other tissues, particularly when large areas and multiple sampling may be required.  相似文献   

4.
Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no "new" components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.  相似文献   

5.
Summary In the present study, we have investigated the applicability of semi-thin sections from low temperature Lowicryl K4M-embedded tissues for cytochemical labelling with protein A—gold and lectin—gold complexes. In order to ensure the best possible signal-to-noise ratio antibodies, protein A—gold and lectin—gold were applied in concentrations used for labelling at the electron microscope level. Furthermore, due to the lack of an appropriate chemical procedure for resin removal, untreated semi-thin sections were incubated. Under such conditions, semi-thin sections displayed either no visible staining or only a faint incomplete staining. However, following photochemical silver reaction, the latent or faint incomplete staining was rendered visible in most cases. It is concluded that the same block of Lowicryl K4M-embedded tissue and the same labelling reagents can be used for both light and electron microscopical cytochemical studies. At the light microscopical level, a high degree of structural and specific staining information is obtained. The reactivity of cellular components with antibodies or lectins is preserved even after years of storage of the blocks or slides containing semi-thin sections.  相似文献   

6.
An in-situ enzyme histochemical method is described that preserves the tissue and cell structure as well as the enzyme activities. Flower buds at different developmental stages from wild-type and transgenic Brassica napus plants, the latter containing the GUS gene under the control of a tapetum-specific promoter, were used as starting material. The method is based on the following principles: processing of the tissue on crushed ice, no fixation but a pretreatment with spermidine, partial dehydration with acetone, and a final embedding in a water-miscible glycol methacrylate resin at 5°C. This method was used to set up a sensitive p-glucuronidase histochemical assay with a high resolution. A succinate dehydrogenase assay was included as a control for tissue and cell viability as well as a standard for the relative metabolic activities between different tissues and cell types.  相似文献   

7.
In the absence of other factors known to influence sectioning properties, high environmental relative humidity is shown to yield poorly embedded tissue. Humidity-related effects are avoided if the following embedding precedure is used: impregnate tissues using the following solutions 1) 70% alcohol—5 minutes, 2) 95% alcohol—4 × 15 minutes, 3) absolute alcohol—3 × 40 minutes, 4) acetone—2 × 15 minutes, 5) 1:1 mixture of acetone-epoxy resin (DDSA, 63.4 g; Araldite 502, 5.6 g; Epon 814,39.4 g; DMP-30, 2.6 g)— 1 hour, 6) acetone-epoxy resin 13—1 hour, 7) epoxy resin—1 hour: complete the preparation of blocks as follows 8) when tissues have been oriented in epoxy resin in flat embedding molds, place molds in one evacuated vacuum desiccator 10 cm above a 2 cm layer of Drierite for 24 hours at room temperature, 9) raise temperature to 60 C and maintain for 3 days to cure resin.  相似文献   

8.
Staining racks, each containing 20 shallow compartments, were constructed by drilling 1.5 cm holes in 8 × 11 cm sheets of 1 mm thick Darvic, an unplasticised polyvinyl-chloride compound, and cementing fibre-glass gauze of 1.3 mm mesh size to one surface. Sections were placed serially—one to each compartment—in the racks, and stacks of up to 9 racks were clamped by Perspex (methyl methacrylate) nuts and bolts, and side clamps. Thus, sections could be handled easily and kept in strict serial order, even in bulk. For Nauta staining, brains had to be gelatin embedded before sectioning. By storing sections in groups of 4 in ice-cube trays, evenly spaced series could be selected for placement in the racks. As many as 160 sections were taken together through all stages of the Nauta method, the timing of critical stages being controlled by taking 4 to 6 free-floating sections, together with the racks, through the various solutions.  相似文献   

9.
Summary The effects of ten fixation protocols on the subsequent binding of eight lectins to various mouse tissue sites have been systematically evaluated. The fixatives used were neutral and buffered formalin—saline, Bouin's fluid, 95% ethanol, Carnoy's fluid, calcium acetate—paraformaldehyde, and mercuric chloride both before and after removal of mercury pigment. These were compared with frozen sections of unfixed tissue and frozen sections post fixed in paraformaldehyde. Lectins used were PNA, DBA, SBA, BPA, UEA 1, GS I, GS II and MPA. Ethanol was found to be the superior fixative, closely followed by mercuric chloride. Paraformaldehyde was a poor fixative of both paraffin and frozen sections. It is recommended that, where a choice is possible, the fixation protocol appropriate to the particular lectin and tissue binding site is selected. Within certain limitations, formalin—saline proved an adequate fixative for the study of routine paraffin-processed tissue sections.  相似文献   

10.
Summary In an attempt to optimize the immunohistochemical procedure for visualizing neuronal markers, such as neuropeptides, in the human skin, different alternatives in all steps of the process were compared. We have studied the influence of type of immunohistochemical method, the biopsy technique, including the size of the punch biopsy, anaesthesia, the choice of fixative and the time of fixation, the storage process, the sectioning parameters, incubation procedure, the type of fluorophore-conjugated antibody and its dilution, mounting and storage, and, finally, microscopical examination.The following procedure was found to give the best result: punch biopsies of 3 mm, taken under local anaesthesia using lidocaine injected into the dermis-subcutis at the place of biopsy; fixation by a buffered 10% formalin solution containing 14% of saturated picric acid for 2 h at 4°C; storage in 10% sucrose buffer for at least 24 h up to 1 month at 4°C or deep-frozen to -70°C for 2 months (with only a minor structural deterioration); cryostat sectioning of the biopsies with a section thickness of 14 m and with the cutting edge perpendicular to the skin surface; rhodamine (TRITC)-conjugated, instead of fluorescein-isothiocyanate (FITC)-conjugated, secondary antiserum, since it gives a lower background fluorescence; and for the incubation and mounting procedures, our standard laboratory routines were applied. The result is an optimal indirect immunofluorescence technique, to be applied in dermatology. We also found that biopsies taken under local anaesthesia with chlorethyl spray lost almost all immunofluorescence for several neuronal markers in the epidermis-upper dermis.  相似文献   

11.
We have developed a method for histochemical demonstration of a wide range of enzymes in freeze-dried, resin-embedded tissue. Freeze-dried tissue specimens were embedded without fixation at low temperature (4 degrees C or -20 degrees C) in glycol methacrylate resin or LR Gold resin. Enzyme activity was optimally preserved by embedding the freeze-dried tissue in glycol methacrylate resin. All enzymes studied (oxidoreductases, esterases, peptidases, and phosphatases), except for glucose-6-phosphatase, were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-drying combined with low-temperature resin embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, high enzyme activity, and excellent tissue morphology.  相似文献   

12.
We describe a method for enzyme histochemical demonstration of NADH dehydrogenase in cold (4 degrees C)-processed resin-embedded tissue. The effects on NADH dehydrogenase activity of processing tissue through a variety of dehydrating agents and embedding in three different acrylic resins were evaluated. The optimal procedure to maintain NADH dehydrogenase activity used a short (3-hr) fixation in 1% paraformaldehyde solution, followed by dehydration in acetone and embedding in glycol methacrylate resin. Embedding of tissue in resin combined preservation and accurate localization of NADH dehydrogenase activity with good tissue morphology. Blocks of the resin-embedded tissue could be stored at room temperature for at least 6 months without loss of NADH dehydrogenase activity.  相似文献   

13.
A sensitive method of histochemical analysis of cellular and tissue glycoconjugates on semithin sections using lectins is suggested. For fixation tissue bioptates were incubated for 4 h in a 2.5% glutaraldehyde in phosphate buffered saline (PBS) at 4 degrees C, then washed for 1 h in 0.2 M glycine in PBS. After epon-araldite embedment and preparation of semithin sections, the resin was removed in saturated ethanol-KOH solution during 5-10 s. Endogenous perooxidase was inactivated in methanol containing 0.3% H2O2. For identification of lectin-binding sites semithin sections were incubated for 30 min in a 0.005% solution of lectin-peroxidase conjugate in PBS and visualized by 0.05% diaminobezidine solution in PBS, containing 0.015% H2O2. The method described ensures good preservation of cellular and tissue glycoconjugates and is highly specific and sensitive.  相似文献   

14.
Summary Glycogen phosphorylase activity has been demonstrated at the ultrastructural level in liver and heart tissue of fasted rats. Unfixed cryostat sections were incubated by mounting them on a semipermeable membrane stretched over a gelled incubation medium. The medium contained a high concentration of glucose 1-phosphate which enables indirect detection of glycogen phosphorylase activity on the basis of the synthesis of glycogen. Tissue fixation, dehydration and embedding for electron microscopical study were performed after the incubation had been completed. The ultrastructure of both liver and heart tissue was rather well preserved. Glycogen granules resulting from glycogen phosphorylase activity were found in the cytoplasmic matrix of both hepatocytes and cardiomyocytes; no relationship with membranous structures could be detected. It is concluded that the semipermeable membrane method is well suited for localizing cytosolic enzyme activities at the ultrastructural level without prior tissue fixation; this opens further perspectives for correlations between histochemical and biochemical data.  相似文献   

15.
Summary In vitro collecting is the process of initiating tissue cultures in the field. In order for in vitro collecting to be broadly available as a technique for collecting plant germplasm, the levels of contamination in such cultures must be controlled. Two techniques for in vitro collecting were compared: leaf punch and needle collecting. The effectiveness of these methods for collecting leaf and stem tissues from plants at tropical and temperature sites was compared. Stem tissue collected by the needle collecting method gave cultures with an average contamination percentage of 31% and 16%, from the tropical and temperate sites, respectively, while with the leaf punch method, average contamination percentages were 90% and 69%. The effectiveness of antimicrobial agents in reducing contamination in leaf punch cultures was evaluated. Addition of the fungicide benlate and the antibiotics cefotaxime and vancomycin, to the leaf punch collections reduced contamination to an average of 30% in the tropical collections and 35% in the temperate collections. Over 90% of both tropical and temperature species collected in multiple samples of 10 or more had at least one clean sample using this medium. The use of either the leaf punch method in combination with a fungicide and antibiotics or the needle collecting technique yielded a high percentage of clean tissues for study and growth.  相似文献   

16.
Fresh tissue slices 2 mm thick and blood films have been fixed by immersion in mercury maintained at 55 C. Fixation is effected in 1.5 min without an excessive temperature rise—the main disadvantage of flaming or hot air used for dry heat fixation. For haematology, in particular, the method is convenient to use as a standard procedure for a fixation that is compatible with many histochemical and most of the usual histological techniques.  相似文献   

17.
Tissue temperature and impedance were measured in dog skin during freezing in situ. The previously frozen skin was removed by punch biopsies 3 days later to permit microscopic evaluation of the extent of necrosis. The histologic observations were related to the temperature and impedance measurements in an effort to determine the usefulness of the monitoring techniques in clinical cryosurgery. Tissue temperature and impedance have a definite relationship in tissue freezing, but the range of temperatures about any impedance values causes some concern. The tissue biopsies showed that an impedance value of at least 10 Mohms is not always associated with tissue death. In these experiments, there was the usual range of temperatures in relation to tissue death, but tissue temperatures of -30 degrees C and colder were always associated with complete necrosis. It is concluded that tissue temperatures are the more accurate and useful monitoring technique to supplement clinical judgment. However, impedance techniques may also be used to monitor therapy, especially if used primarily to monitor depth of therapy, and if controlled by clinical judgment wary of the inaccuracy of the technique.  相似文献   

18.
Summary When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment toUlex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.  相似文献   

19.
Summary Various procedures for nonpolar and polar resin embedment were applied to mouse and rat livers for the study of postembedment immunolocalization of alpha1-fetoprotein, albumin and the microsomal enzyme epoxide hydrolase. Fixations with formaldehyde and with formaldehyde-glutaraldehyde mixtures were used for tissue stabilization. Both fixation schedules did not abolish immunoreactivity. Treatment of liver with inert compounds such as polyvinylpyrrolidones or chemical modification of antigens with ethyl acetimidate prior to embedment improved immuno-staining. Either the low-polarity solvent ethanol or the highly polar ethylene glycol could be employed as dehydrating agents. Antigens were readily localized in sections from Epon 812 embedded livers. For this purpose, polymerized resin had to be partially removed. On the other hand, immunoreactivity of antigens was only faint after embedment in an epoxy resin based on diepoxide octane. Also, antigens reacted faintly in sections from livers which were embedded at 0° C in the polar acrylate-methacrylate based Lowicryl K4M resin. The indirect peroxidase labelled antibody method was as specific and sensitive as the PAP technique. Optimal antigen detection was attained with antibodies isolated by affinity chromatography and purified peroxidase conjugates. Apart from purified immunological reagents, the addition of high molarity sodium chloride and bovine serum albumin to the wash solutions enhanced immunohistological specificity.Supported by the Deutsche Forschungsgemeinschaft (Ku 257/3) Bonn, Federal Republic of Germany  相似文献   

20.
Type II and III fibrillar collagens were localized by immunogold electron microscopy in resin sections of human femoral articular cartilage taken from the upper radial zone in specimens from patients with osteoarthritis. Tissue samples stabilized by high-pressure cryofixation were processed by freeze-substitution, either in acetone containing osmium or in methanol without chemical fixatives, before embedding in epoxy or Lowicryl resin, respectively. Ultrastructural preservation was superior with osmium-acetone, although it was not possible to localize collagens by this method. In contrast, in tissue prepared by low-temperature methods without chemical fixation, collagens were successfully localized with mono- or polyclonal antibodies to the helical (Types II and III) and amino-propeptide (Type III procollagen) domains of the molecule. Dual localization using secondary antibodies labeled with 5- or 10-nm gold particles demonstrated the presence of Types II and III collagen associated within single periodic banded fibrils. Collagen fibrils in articular cartilage are understood to be heteropolymers mainly of Types II, IX, and XI collagen. Our observations provide further evidence for the complexity of these assemblies, with the potential for interactions between at least 11 distinct collagen types as well as several noncollagenous components of the extracellular matrix.  相似文献   

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