Detection of apoptosis and changes in mitochondrial membrane potential with chloromethyl-X-rosamine.
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Apoptosis↗
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Current methods for brow fixation: are they safe?
BACKGROUND: No single technique for fixation of the scalp after forehead-lift is universally accepted. Complications such as alopecia, loss of elevation, implant palpability, paresthesia, and dural injury are possible with the variety of techniques used currently. This anatomic study was designed to evaluate the thickness of the calvarium at selected points used in brow fixation. The depth of cranial penetration necessary for currently used techniques is measured and compared. METHODS: In a study of 14 fresh adult cadavers, calvarial thickness was measured at selected points (A-F) used in various brow-lifting procedures. This was accomplished by drilling holes in selected points and using a depth gauge to measure thickness. Immediately adjacent to selected points, the cranium is prepared for brow fixation using the following techniques: cortical tunnels, 2.0-mm screw fixation (10, 12, and 14 mm), the Mitek 2.0-mm Quickanchor screw, and the Endotine 3.5 Forehead Device. The depths required for adequate fixation and the potential for cranial penetration through the inner table with all the standard techniques are compared. RESULTS: Depth analysis by mean values showed that sites posterior to the coronal suture (points C-F) were thickest. Depth analysis of sites stratified by gender showed that mean values for the thickness of female skulls were greater than those for males. A review of fixation methods found that cortical tunnels at 45 degrees angles never penetrated the inner table in any of the 14 skulls. Mitek screws never penetrated the inner table, and one Endotine post penetrated the inner table on the left side of one cadaver skull. After placement of 10-, 12-, and 14-mm miniscrews at each of the sites, it was found that three penetrated the inner table. The penetrations all were at far lateral sites, posterior to the coronal suture. CONCLUSION: Variation in skull thickness exists among cadaver specimens at different sites on the skull. In this study, thickness increased medially and posteriorly. Women tended to have thicker skulls than men, and age was not a major variable. This is consistent with findings in previous work. Given the unpublished reports of inner table penetration, with cerebrospinal fluid leak after invasive brow fixation, it behooves the surgeon to keep in mind the anatomy of the calvarium and its nuances.
Aged↗
[A new technique for foetal brain fixation and extraction].
We performed a new technique for foetal brain fixation and extraction that offers a remarkable reduction in sampling time facilitates specimen handling while conserving high quality. With the new fixation method, it is possible to obtain samples adequate for macroscopic and microscopic observations and immunohistochemical analysis. The technique involves the creation of an ex vacuo phenomenon in the subarachnoideal space prior to injecting the fixative solution. In this manner, the solution is homogenously distributed. Ease, reproducibility and the possibility of standardizing the procedure are the principal advantages. Low costs, reduced wording time and less need for human resources are other advantages. Histologically, we obtained, quickly, high quality slides with routine and immunohistochemical stains. Disadvantages of the technique derive from the use of formaldehyde and glacial acetic acid, rather than water to wash the samples; thus one must work in a well ventilated area, with gloves, protective glasses and an adequate lab coat to avoid skin and respiratory tract damage.
Brain↗
Extraction of cellular DNA from human cells and tissues fixed in ethanol.
DNA can be extracted from ethanol-fixed lymphoid cells and tissues. The fixation procedure is simple and rapid, and the DNA extraction itself is the same as that normally used for fresh tissue or cells. DNA extracted from ethanol-fixed material is indistinguishable from DNA extracted from fresh samples based on its purity, its ability to be digested with restriction endonucleases, and its ability to specifically hybridize to DNA probes. The capability to extract DNA from ethanol-fixed cells and tissues eliminates the need for stringent handling and storage requirements of fresh or frozen specimens.
DNA↗
A Golgi impregnation technique for thin brain slices maintained in vitro.
A technique for using routine rapid Golgi impregnation procedures on very thin freshly fixed slices (less than 0.5 mm) of brain tissue is described. The technique was particularly successful with hippocampal slices that were maintained and stimulated in vitro prior to fixation. Thin tissue slices were surrounded by thicker sections of tissue to form a 5 mm thick bundle. The tissue bundle was then processed by a rapid Golgi procedure, 5 days each in chromate osmium and silver nitrate solutions. At the end of this time the thin tissue slices were unwrapped from their thicker protecting tissue sections, embedded in celloidin, cut at 60-100 micrometer thickness on a sliding microtome and mounted in permount under cover glass. Qualitative light microscopic analysis of the rapid Golgi impregnated slices revealed fully impregnated cell bodies, dendrites, dentritic spines, axons and axonal varicosities with minimal background artifact. In contrast, unprotected thin tissue slices showed only a dense black artifact without cells or processes.
Animals↗
Evaluation of leukocyte stabilisation in TransFix-treated blood samples by flow cytometry and transmission electron microscopy.
In this report, we have evaluated the effects of a TransFix-based stabilisation technique on leukocyte scatter characteristics, immunophenotyping, membrane permeability, absolute cell counting and morphology to extend previously reported flow cytometric data focused on the lymphocyte population. We show that scatter characteristics, immunophenotyping and absolute cell counting are well preserved, particularly in the lymphocyte population. Nevertheless, a general increase in membrane permeability, evaluated by propidium iodide (PI) uptake, was observed in TransFix-treated leukocyte subsets. Ultrastructural observations show selective morphological preservation (up to 10 days of storage) of lymphocytes and, to a lesser extent, of monocytes. In contrast, granulocytes have necrosis-like features, although the plasma membrane seems well preserved. Therefore, electron microscopy observations reflect modifications induced in different cell populations as evidenced by flow cytometry (FC). The data indicate that this short-term stabilisation method is particularly suitable for the analysis of human lymphocytes and it is a good procedure for quality control programmes for inter- and intra-laboratory performance evaluation; good results are obtained with respect to antigen definition and absolute cell counting procedures. Any apoptotic pathways in leukocyte subsets are blocked for at least 10 days.
Adult↗
Physical properties of a porcine internal thoracic artery fixed with an epoxy compound.
This study aimed to investigate the physical properties of a porcine internal thoracic artery fixed with an epoxy compound. Fresh and glutaraldehyde-fixed porcine arteries were used as controls. Both the epoxy-fixed and glutaraldehyde-fixed porcine arteries showed significant increases in fixation index and denaturation temperature as compared to the fresh one. Additionally, the resistance of porcine arteries against bacterial collagenase in vitro degradation increased significantly after cross-linking with the epoxy compound of glutaraldehyde. The reduction of the tensile strength of the epoxy-fixed artery after bacterial collagenase degradation was more severe than in its glutaraldehyde-fixed counterpart. Generally, with increasing pH, temperature or fixative concentration, the fixation indices and denaturation temperatures of the epoxy-fixed porcine arteries increased. The results obtained in this study may be used to optimize the fixation process for developing a small-diameter biological vascular graft fixed by an epoxy compound.
Animals↗
Marking the surgical margins of specimens: commercial acrylic pigments are reliable, rapid and safe.
AIM: To find a medium to mark the surgical margins and orientation of complex specimens that is reliable, easy to use, safe and inexpensive. METHODS: Several different paints, dyes and pigments were tested. A series of pigments supplied by SICPA Australia Pty Ltd, when fixed to the specimen in acetic formalin, was used on over 7000 complex specimens. RESULTS: The pigments were easily visualised macroscopically on gross specimen and processed blocks and microscopically at the surgical margins. Review of early material confirmed its stability. CONCLUSION: These pigments fulfill all the criteria of being reliable, easy to use, inexpensive, safe and stable and have been used successfully in our department for 4 years.
Acrylates↗
HistoChoice as an alternative to formalin fixation of undecalcified bone specimens.
We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visualized more easily in HistoChoice fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.
Animals↗
Effects of different fixatives on beta-galactosidase activity.
beta-Galactosidase (beta-Gal) staining is widely used to demonstrate specific gene expression during evaluation of gene targets in vivo. This technique is extremely sensitive to fixation. Optimal fixation conditions are necessary to obtain the maximal beta-Gal activity. In this experiment, Carnoy's and three different aldehyde fixatives were used at different temperatures and over different time points. Kidneys from LacZ-stop-human alkaline phosphatase (ZA/P) double reporter mice were used to generate positive material for the experiment. The results show that glutaraldehyde combinative solution (LacZ) produced the most consistent and reliable results. Paraformaldehyde and formaldehyde were effective as fixatives only at 4C for a period of less than 4 hr, and Carnoy's solution destroyed beta-Gal activity.
Animals↗
Immunogold electron microscopic localization of timothy grass (Phleum pratense) pollen major allergens Phl p I and Phl p V after anhydrous fixation in acrolein vapor.
We used the vapor phase of acrolein as an anhydrous fixative for timothy grass pollen in an immunogold double-labeling localization study of two different major allergens, Phl p I and Phl p V. More than 48 hr of fixation were needed for the subcellular pollen structures to be satisfactorily stabilized. The immunoreactivity of acrolein-fixed pollen allergens was not destroyed even after prolonged acrolein fixation. By immunoblotting, the two allergens differ in their immunological and structural characteristics. Electron microscopic localization traced the allergens at least partially to different subcellular pollen compartments.
Acrolein↗
Heat-induced antigen retrieval: what are we retrieving?
Of all molecular aspects lost and recovered during formalin fixation and antigen retrieval, respectively, electrostatic charges have probably received the least attention. This review will focus on our work during the past 7 years. It strongly supports the tenet that electrostatic charges, i.e., net negative on antigens and net positive on antibodies, play an important part in immune reactions and therefore should be given greater attention when focusing on quality control in immunohistochemistry.
Antibodies↗
Preparatory tests for immunodetection of microtubules in protoplasts during IML-2.
Preparatory tests to improve methods for detection of cortical microtubules (cMTs) in rapeseed protoplasts for the IML-2 experiment "TRANSFORM" were undertaken. This study is based on the results obtained in the "PROTO" experiment onboard the shuttle Discovery during an 8-day IML-1 flight in 1992. The use of free-floating protoplasts on IML-1 made it technically impossible for the astronauts to remove the glutaraldehyde fixative during the orbital period resulting in high background fluorescence which made it very difficult to detect MTs. In order to avoid this on the IML-2 mission, protoplasts will be immobilized in alginate beads. The effect of variable concentrations and fixation periods for two different fixatives on the preservation of cMTs was tested. Best results were obtained using 3.5% paraformaldehyde (PFA) for 1 hour, but 1% glutaraldehyde (GA) also gave acceptable results. The effect of low temperatures on microtubule depolymerization was also examined as freshly isolated protoplasts have to be kept at 5 degrees C for up to 20 hours pre-launch and before activation of Biorack. Only slight changes in cMT-appearance were observed at 4 degrees C indicating a minor depolymerization compared to the cMTs in non-chilled protoplasts.
Alginates↗
Scanning electron microscopy of the forming enamel of rat incisor: influence of fixative and treatments interacting with the organic matrix.
The forming enamel of rat incisor was either treated before fixation with one of these five treatments: ultrasonication, NaOCl, chloroform/methanol, NaOH or tween 20; or the incisors were first fixed and then submitted to one of these treatments. The structure of the forming enamel, compared with sections fixed with aldehyde and submitted to osmic maceration, depends on the way the samples were treated and on the fixative used. An amorphous material, removed by osmium tetroxide but not by aldehyde solution was seen to be located between fiber-like structures. In the region of inner enamel secretion the thickness of interrod septa was fairly constant, whereas in the region of outer enamel secretion, transverse septa of interrod enamel gradually became thinner and finally disappeared. The longitudinal septa did not display such changes. Longitudinal grooves and ridges were seen at the end of enamel completion. These longitudinal septa finally merged and formed a flat surface.
Animals↗
Quick bacterial microwave fixation technique for scanning electron microscopy.
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Bacteria↗
Advances in high-pressure and plunge-freeze fixation.
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Cryopreservation↗
A molecular mechanism of formalin fixation and antigen retrieval.
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Amino Acids↗
Scanning electron microscopy of the luminal epithelium of the mouse uterus.
Four methods of manipulating mouse uterine tissue during fixation, preparatory to examination of the luminal epithelium by scanning electron microscopy (S.E.M.), are described and the results assessed. The appearance of the epithelium varies according to the technique used, the choice of method depending on the type of information required from the S.E.M. study. Surface topography is preserved in a condition most closely resembling that of fresh tissue by opening the uterus and fixing the tissue flat. Critical examination of cellular and subcellular surface detail, however, depends on adequate spreading of the epithelial layer during fixation by distension of the uterine lumen under positive pressure. The method of tissue manipulation has a more profound effect on the appearance of the sample than any of the dehydration techniques which follow. For routine S.E.M. examinations at medium magnifications (smaller than X10,000), specimens which were air dried from alcohol, acetone or ether, or freeze dried, gave satisfactory results. Air drying from amyl acetate, and CO2 critical-point drying gave superior results at higher magnifications (greater than X10,000) with better preservation of individual microvilli.
Air↗