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PubMed1952

A study of fixation for electron microscopy.

Osmium tetroxide fixation of tissue blocks, as usually effected, is preceded by an acidification of the tissue. This acidification is probably responsible for morphological alterations which are notably disturbing in electron microscopy. The acidification and the resulting morphological alterations cannot be prevented by homogenizing the tissue directly in OsO(4) solutions or by adding enzyme inhibitors (fluoride, iodoscetamide) to the fixative. Fixation experiments with buffered OsO(4) solutions have shown that the appearance of the fixed cells is conditioned by the pH of the fixative. The quality of fixation can be materially improved by buffering the OsO(4) solutions at pH 7.3-7.5, The acetate-veronal buffer appeared to be the most favorable of the buffers tested, Because of these findings, 1 per cent OsO(4) buffered at pH 7.3-7.5 with acetate-veronal buffer is recommended as an appropriate fixative for electron microscopy.

Electrons↗
PubMed2001

Alkaline fixation-resistant acid phosphatases in human tissues: histochemical evidence for a new type of acid phosphatase in endothelial, endometrial and neuronal sites.

The effect of pH during formalin fixation on acid phosphatases in human tissues was studied. Lysosomal-type acid phosphatase was sensitive to alkaline fixation, being completely inactive after fixation at pH 9.0. Prostatic and tartrate-resistant osteoclastic/macrophagic types were alkaline fixation-resistant, as was an acid phosphatase localized in endothelium, endometrial stromal cells and intestinal nerves. The latter activity was further separable into fluoride- and tartrate-sensitive beta-glycerophosphatase and fluoride-sensitive, tartrate-resistant alpha-naphthyl phosphatase. The activities appeared to represent either different, tightly associated enzymes or separate activity centres of a single enzyme. Alkaline fixation-resistant alpha-naphthyl phosphatase at endothelial, endometrial and neuronal sites was also well demonstrated in unfixed or neutral formalin-fixed sections as tartrate-resistant activity similar to classical tartrate-resistant acid phosphatase, but these phosphatases appear to be antigenically different. Alkaline fixation-resistant acid phosphatase showed a restricted tissue distribution both in endothelium (mainly in vessels of abdominal organs) and at neuronal sites (only in intestinal nerves). Alkaline fixation-resistant acid phosphatase appears to represent a previously unknown or uncharacterized enzyme activity whose chemical properties could not be classified as any previously known type of acid or other phosphatases.

Acid Phosphatase↗
PubMed1990

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes.

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.

Animals↗
PubMedDate not supplied

The effect of screw insertion torque on tendons fixed with spiked washers.

The long-term success of a hamstring tendon graft depends not only on the type of device that is used for fixation but also on the mechanical interlocking of the soft tissue between the fixation device and bone. The purpose of this study was to evaluate the effect of screw insertion torque on the structural properties of soft tissue fixed to bone with a spiked metal washer. Two bovine tendons, one similar in size to a human semitendinosus tendon and the other similar in size to a human gracilis tendon, were secured to a bovine femur using a figure-of-8 technique with screws and metal spiked washers. A single load to failure was applied at 25 mm/sec. A significant positive linear correlation was observed between fixation screw insertion torque magnitude and the ultimate failure load value. An increase in the fixation screw insertion torque produced an increase in the ultimate failure load value. Similarly, there was a significant positive linear correlation between fixation screw insertion torque magnitude and the average maximum linear load value. No relationship was detected between screw insertion torque magnitude and the linear stiffness values of the tendon-fixation construct, indicating that a reproducible model was used. This study demonstrates that screw insertion torque is an important variable that controls the initial strength of soft tissue fixation to bone.

Animals↗
PubMed1991

The small pin circular fixator for proximal tibial fractures with soft tissue compromise.

We used a Monticelli-Spinelli small pin circular external fixator in five cases, in combination with closed reduction or limited open reduction internal fixation, to salvage a satisfactory result in juxtaarticular, intraarticular fractures of the proximal tibia, when associated soft tissue compromise prevented standard fixation with plates and screws. The small pin circular fixator allows juxtaarticular placement of the small pins, enhancing stabilization of the comminuted fractures, allowing early range of motion of the joint and early patient mobilization. The small diameter pins support the soft cancellous bone fragments. This technique attempts to combine the benefits of traction, external fixation, and limited internal fixation. We recommend this method as a salvage procedure when plates and screws are contraindicated because of poor bone and soft tissue conditions.

Adult↗
PubMed1995

Field emission SEM comparison of four postfixation drying techniques for human dentin.

Critical-point drying (CPD) is generally considered essential for the preparation of biologic specimens for electron microscopy. Several attempts have been made to introduce alternative techniques. More recently, this problem has arisen in dentistry, because of the new developments in dentin bonding. The present study focuses on three alternative techniques to CPD: hexamethyldisilazane (HMDS) drying, Peldri II drying, and air drying. Twenty-four dentin disks were obtained from noncarious extracted human molars by microtome sectioning parallel to the occlusal surface. The dentin surfaces were etched with polymer-thickened, silica-free, 10% phosphoric acid semigel, fixed, dehydrated, and dried with one of the four techniques. The specimens were observed in two perpendicular planes, showing dentinal tubules in longitudinal view and cross-section, using a field emission scanning electron microscope. The intertubular demineralized dentin zone was composed of three different successive layers, which did not substantially differ between CPD and Peldri II drying, but were more evident in HMDS-dried specimens: first, an upper layer of denatured collagen and residual smear layer particles, with sectioned collagen fibrils and few open intertubular pores; second, an intermediate layer of closely packed cross-sectioned collagen fibers; and third, a deeper layer with unfilled spaces, scattered hydroxyapatite crystals, and few collagen fibers. HMDS drying seemed to preserve better the collagen network and the microporosity of the demineralized dentin surface. Moreover, HMDS drying is easy to perform. The air-drying method caused some artefacts, such as surface collapsing and thickening of the denatured collagen layer.

Air↗
PubMed1990

Improved cryoprotection and freeze-substitution of embryonic quail retina: a TEM study on ultrastructural preservation.

Conditions for cryofixation and freeze-substitution crucial to the ultrastructural preservation of embryonic quail retina were improved. As freeze-substitution makes gentle dehydration and chemical fixation of tissue possible, the suitability of different cryoprotectants were tested in the preceding cryofixation. Additionally, different conditions for chemical prefixation were studied. In cryofixation, all of the "classic" cryoprotectants caused more or less severe tissue destruction. Only dimethylformamide (DMF) and--with certain reservations--dimethylsulfoxide (DMSO) yielded improved structure preservation. Perfusion fixation with a mixture of formaldehyde/glutaraldehyde (FA/GA) was superior to GA alone. In comparison to conventional fixation and dehydration methods, freeze-substitution yielded better ultrastructural preservation of the embryos with fewer artifacts.

Animals↗
PubMed1994

Use of fluorescent europium chelates as labels in microscopy allows glutaraldehyde fixation and permanent mounting and leads to reduced autofluorescence and good long-term stability.

In the present study europium chelates were introduced as alternative fluorescent labels for microscopy and their effect on enhanced autofluorescence caused by the glutaraldehyde fixative was investigated. Glutaraldehyde fixation was used to stabilize the cells for a permanent mount after the immunocytochemical reaction. The europium signal in time-resolved fluorescence microscopy was shown to be free of autofluorescence when strong cross-linking fixation with glutaraldehyde was used and the signal-to-background ratio obtained was 2,400 or better. It was also shown that the europium signal was stable in daylight and at room temperature. Fluorescent europium chelate used in this experiment provides excellent contrast and long-term stability for the samples with glutaraldehyde fixation and permanent mounting.

Antibodies, Monoclonal↗
PubMed2005

Comparison of cell fixation methods of induced sputum specimens: an immunocytochemical analysis.

Induced sputum (IS) is a non-invasive method to evaluate airway inflammation. Various techniques are used to fix IS cells but their respective value has never been compared. We aimed to determine the best IS cell fixation technique for cellular markers staining. Cells were fixed using four methods: 1) periodate-paraformaldehyde-lysine (PLP)-sucrose, 2) paraformaldehyde 4% on slide and 3) in solution and 4) acetone-methanol. Analysis was based on percentage of positive cells compared to total cell counts stained by hematoxylin and quality of staining. Using PLP-sucrose resulted in a higher percentage of positive cells for CD3 and a better quality of staining. Acetone-methanol showed a lower percentage of positive cells for CD68 and a poor quality. PLP-sucrose gives the best results for the preservation of the studied cell markers and acetone-methanol the worst.

Adult↗
PubMed1998

Optimization of the immunohistochemical demonstration of keratins in paraffin wax-embedded mouse skin.

The purpose of this study was to develop an immunoperoxidase technique for the detection of cytokeratins in samples of paraffin wax-embedded adult and fetal skin from NMRI mice, with various antibodies (Troma-1, LL001, 8.60, MCK5, MCK6, AF129) that have been tested mainly on fresh-frozen sections. Each antibody was tested with three different fixatives (10% neutral buffered formalin, Bouin's fluid, and 70% ethanol) and two distinct pretreatments (enzymatic digestion with trypsin, or heat treatment). The best results, in terms of non-specific background labelling, morphological preservation and intensity of specific labelling, were obtained (1) for adult skin, by the use of Bouin's fluid, heat pretreatment and antibodies LL001, MCK5, MCK6 or AF129, and (2) for fetal skin, by the use of 70% ethanol, heat pretreatment and antibody Troma-1. Monoclonal antibody 8.60 gave the best results when the use of 70% ethanol was combined with either enzymatic digestion or heat pretreatment.

Animals↗
PubMed1997

A new RNA-friendly fixative for the preservation of penaeid shrimp samples for virological detection using cDNA genomic probes.

In situ hybridization analysis of shrimp histological sections, utilizing Taura syndrome virus (TSV) specific cDNA probes, is the most sensitive diagnostic technique presently available for the detection of this penaeid shrimp viral disease. However, false negative genomic probe results are obtained frequently from samples of Pacific white shrimp, Penaeus vannamei, that have been preserved with Davidson's AFA (acetic acid, formaldehyde, alcohol) fixative and that, otherwise, demonstrate pathognomonic TSV lesions by routine histology. This problem was linked to prolonged storage of shrimp samples in Davidson's fixative, which is highly acidic (pH approximately 3.5-4). Degradation of TSV genomic RNA was hypothesized to be due to either fixative- induced acid hydrolysis and/or acidophilic endogenous ribonuclease activity. Routine H and E histology and in situ hybridization analyses were conducted on equal numbers of TSV infected P. vannamei juveniles that were preserved for four different time periods (2, 6, 10 and 14 days) with either Davidson's fixative or a new, near neutral (pH approximately 6.0-7.0), RNA-friendly fixative (R-F) that was developed by the authors. In situ hybridization assays were conducted with and without R Nase precautions and all of the samples tested contained moderate to severe TSV lesions by routine histology. Davidson's preserved samples produced weak TSV probe signals after 2 days fixation, but did not react with the probes in those samples that were stored for > 6 days in the fixative. In contrast, TSV was detectable by gene probe in all of the time treatment samples preserved with the new R-F fixative. Equivalent in situ hybridization results were obtained when the same samples were analyzed in the absence of RNase-free conditions. These findings suggest that TSV RNA is degraded when samples are stored in an acidic fixative, such as Davidson's, for more than 2 days and that this problem can be prevented through preservation of shrimp samples with R-F fixative. The efficacy of this new fixative is demonstrated and the results show that RNase-free conditions are not necessary for conducting TSV in situ hybridization analyses.

Animals↗
PubMed2000

Recovering DNA and optimizing PCR conditions from microdissected formalin-fixed and paraffin-embedded materials.

Microdissection is a powerful technique in molecular pathology and genetic investigations. To detect genetic alterations such as gene mutation or deletion from tumor specimen, the purity of target cells is extremely critical. Unwanted cell contamination will dramatically dilute the detectable level of the abnormality. The major obstacle in clinical research is to obtain sufficient and qualified DNA from a small amount of formalin-fixed and paraffin-embedded materials. We have successfully modified our previous protocols and overcome the difficulties of recovery of DNA. After these modifications, almost every single formalin-fixed and paraffin-embedded specimen has been successfully amplified in the required DNA region.

DNA↗
PubMed2003

Alternative method of rapid drying vascular specimens for scanning electron microscopy.

PURPOSE: To report the use of hexamethyldisilazane (HMDS) as an alternative to critical point drying for preparing stented canine peripheral vessels for scanning electron microscopy (SEM). TECHNIQUE: Vascular specimens were fixed in 4% formaldehyde overnight, dehydrated in a graded ethanol series, followed by immersion in 100% hexamethyldisilazane. After air drying, the specimens were mounted on stainless steel stubs, coated with gold, and examined in the SEM. The electron micrographs were of high quality, showing the layers of the vascular wall and the incorporated stent covered by a neointimal layer. The micrographs were comparable to corresponding histological sections, but detailed endothelial patterns were more visible. CONCLUSIONS: HMDS treatment and subsequent air drying provides good quality scanning electron micrographs that reveal both endothelial patterns and the layered architecture of stented vessels. The disadvantage of HMDS drying may be a shrinkage and distortion similar to other drying agents. Ease of handling, low cost, and a high rate of success are advantages that favor HMDS desiccation over other drying methods.

Animals↗
PubMed1994

A comparison of heating modes in rapid fixation techniques for electron microscopy.

Recent studies have established that microwave irradiation during aldehyde fixation of electron microscopy specimens can reduce fixation times substantially. Similar reductions in duration of histochemical and light microscopy procedures have been reported. Both thermal and non-thermal effects of microwaves have been proposed to explain these dramatic decreases in processing time. Possible thermal effects include increases in fixative diffusion and reaction rates and increased formation of glutaraldehyde monomers. Proposed non-thermal effects include preferential orientation of fixative molecules by the microwave field and other more speculative direct microwave effects. Several reported attempts to produce rapid fixation without temperature increase by cooling specimens during irradiation have produced conflicting results. If rapid fixation is a thermal effect, other heating modes in addition to microwave exposure should produce similar effects. We show that for mouse liver samples (< or = 1 mm3) comparable fixation can be obtained with microwave irradiation, conductive and convective heating in a waterbath, and resistive heating with a low-frequency (1 kHz) current passed through the fixative solution. We also show that using an efficient convective cooling method to prevent temperature increase during microwave exposure produces unsatisfactory fixation. These results are consistent with thermal mechanisms for rapid fixation.

Animals↗
PubMed1985

Immunoelectron microscopic localization of glomerular IgA deposits using a protein A-colloidal gold probe.

Currently there is increasing interest in the localization and accurate determination of the composition of protein deposits involved in the pathogenesis of human glomerulonephritis using immunoelectron microscopy. Previous studies have largely relied on capricious immunoperoxidase techniques applying both the preembedding and postembedding methods to renal tissue. Both methods identify a single antigen, but preservation of ultrastructure is suboptimal, and there is the potential problem of imprecise localization of antigen due to diffusion of the peroxidase reaction product. A less demanding method of ultrastructural antigen identification for renal tissue has been employed with success in clearly localizing glomerular deposits of IgA. The technique involves fixation of tissue in periodate-lysine-paraformaldehyde, embedding in L. R. White resin, and labeling by an indirect immunologic reaction using protein A-gold as an electron-dense marker. This technique provides an accurate marker for the localization of glomerular antigen while retaining optimal tissue morphology, and we predict that this method will have wide application in human and experimental renal disease.

Glomerulonephritis↗
PubMed1994

A study of criteria permitting the use of plastinated specimens for light and electron microscopy.

Plastination permits the preservation of anatomical specimens in a physical state approaching that of the living condition. We studied the possibility of using silicone plastinated fragments of spleen and pancreas for optical and electron microscopy, and found that with an adequate fixation protocol, plastinated specimens can be used for both light microscopy and ultrastructural studies. Deplastination with sodium methoxide permitted production of clean sections. Artifacts produced by plastination/deplastination could be nearly eliminated by glutaraldehyde/formaldehyde fixation. The (Biodur) silicone S10 polymer is transparent and stable in an electron beam, and plastinated tissues can be contrasted or colored similar to tissues embedded in Epon 812. In addition to being very life-like, plastinated tissues are stable and easy to handle. They can also be used for electron and light microscopic studies. This technique may also allow retrospective epidemiological studies of archived pathology specimens.

Animals↗