IBF as a formalin substitute fixative in prostate biopsy pathology.
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Biopsy↗
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The pore density in the inner wall endothelium of Schlemm's canal of glaucomatous eyes.
PURPOSE: In a prior study, it has been reported that glaucomatous eyes have a significantly lower density of pores in the inner wall of Schlemm's canal than do normal eyes. However, in that study the glaucomatous eyes were fixed at much lower flow rates than the normal eyes, and that is now known to affect inner wall pore density. The objective of the current study was to compare the inner wall's pore density in glaucomatous and normal eyes, accounting for the effects of fixation conditions. METHODS: Outflow facility was measured in enucleated glaucomatous human eyes. Eyes were fixed under constant flow conditions, microdissected to expose the inner wall of Schlemm's canal, and prepared for scanning electron microscopy (SEM). The density and diameter of the two subpopulations of pores in the inner wall, intracellular and intercellular (or "border") pores, were measured. Data were compared with those in previous studies of normal eyes. RESULTS: As previously reported, pore density decreased with increasing postmortem time and increased with increasing volume of fixative passed through the outflow pathway and with increasing fixation time. Linear regression analysis indicated that glaucomatous eyes had less than one fifth the number of pores than normal eyes have, after accounting for the influence of volume of fixative perfused through the eyes (835 pores/mm(2) in normal eyes versus 160 pores/mm(2) in glaucomatous eyes). A nonlinear regression of pore density versus fixative volume produced a pore density at zero fixative volume that was not statistically different from zero. If true, this implies that all (or nearly all) inner wall pores observed by SEM are fixation artifacts. The density of intracellular pores and the diameter of these pores correlated with the density and diameter of the border pores, respectively. CONCLUSIONS: Inner wall pores are reduced in glaucomatous eyes. If pores are physiological structures, the elevated intraocular pressure characteristic of glaucoma may be explained by decreased porosity of the inner wall endothelium. Both border and intracellular pores seem to be induced in a similar fashion by fixation. The unlikely possibility that all inner wall pores are fixation-induced cannot be excluded. If so, a fundamental reassessment of the mechanism by which aqueous humor crosses the inner wall endothelium is necessary.
Aged↗
[Staining of bone canaliculi using decalcified bone tissues].
Recently we were able to stain bone canaliculi using the decalcified bone tissues, according to a modified method of the Bodian staining method, which is commonly used for staining the nerve fibers with silver impregnation of protargol. The bone tissues from normal rabbits, rats, and humans were decalcified with buffered EDTA solution after 10% formalin fixation. The tissues were embedded in paraffin and were sliced at 4 microns then the sections were attached to the gelatin-coated glass slides. These sections were treated first with potassium dichromate followed by 2% protargol solution containing copper. After gold chrolide treatment, they were immersed in oxalic acid. Both the lacunae and canaliculi of osteocytes were stained black in sharp contrast to bone matrix of red purple. Using the present staining method, the bone canaliculi were clearly detectable in fine details without producing apparent artificial damage of soft tissues. The canaliculi showed also markedly complex fine structures. By combining the present staining method with eosin or toluidine blue staining, we were able to recognize even immature osteocytes in enchondral ossification at growth cartilage with fine bone canaliculi. From these results, we conclude that the present method is very useful in histopathological studies of the lacuna-canalicular system of osteocytes in the decalcified bone tissues.
Animals↗
Ring-stabilization technique for collection of avian air sacs.
Histologic features of chicken air-sac membranes harvested with rings were compared with those of membranes collected on cards. Ring stabilization of the tissues throughout fixation, dehydration, and embedment was found to reduce markedly the artifacts and the loss of normal tissue architecture observed within the wall of card-collected air sacs.
Air Sacs↗
Handling of large brain specimens: the use of microwaves. A review.
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Brain↗
Breast biopsy specimen fixation.
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Biopsy↗
Cochlear implant fixation with ionomeric bone glue.
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Bone Cements↗
gamma-Glutamyl transpeptidase demonstrated in tissue sections embedded in glycol methacrylate resin.
A method is described for the histochemical demonstration of gamma-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 degrees C prior to embedding at 4 degrees 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.
Acrylates↗
Accessibility of 7-aminoactinomycin D to lymphocyte nuclei after paraformaldehyde fixation.
DNA-protein interactions, mainly DNA-histone interactions, are thought to play an essential role in the packing mechanisms of chromatin as well as in transcriptional control. In this context it is important to develop new methods to study DNA-protein interactions and structural changes associated with them. Paraformaldehyde (PFA) has been shown to crosslink proteins, mainly histones, to DNA; and in this short study we have used a system with the DNA-binding dye 7-aminoactinomycin D (7-AAMD) as an indirect probe for PFA fixation. The aim was to investigate the dynamics of fixation on the binding of this dye to intact human lymphocytes. The results show a decrease in accessibility of 7-AAMD,initially affecting both the nonspecific binding of 7-AAMD and the high affinity binding sites, and thereafter mainly the high affinity binding sites. We conclude that fixation with PFA is a long-term reaction that requires a fixation time of several hours at a sufficient concentration to be completed. Our findings suggest that staining with a low concentration of 7-AAMD after extensive PFA fixation may be used to obtain information on DNA-protein interactions in intact cells.
Binding Sites↗
Time-resolved fluorescence imaging for quantitative histochemistry using lanthanide chelates in nanoparticles and conjugated to monoclonal antibodies.
Tissue and cell examinations have a potential to produce extremely valuable information about antigen quantities in samples. Using currently available methods, a truly quantitative analysis is nearly impossible. We have previously shown that immunohistochemical (IHC) detection of prostate-specific antigen and human glandular kallikrein from prostatic tissue, together with time-resolved fluorescence imaging (TRFI), is a suitable method for obtaining quantitative data from biological samples and that the signal response is linear. In this paper we show that Eu-chelate containing particles in the nanometer range are suitable labels for quantitative IHC. Even single nanoparticle molecules can be detected by TRFI and the signals measured can be readily quantitated. The signal intensity correlates very well with the amount of bound label, and the use of nanoparticles could markedly improve the sensitivity of quantitative IHC methods. TRFI provides a powerful tool for providing quantitative data about antigens or transcripts in tissue sections or cultured cells. It is also of major importance in standardization and optimization of protocols for fixation and tissue preparation, including antigen retrieval methods.
Animals↗
Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry.
BACKGROUND: Immunophenotyping of whole blood (WB) and isolated peripheral blood mononuclear cells (PBMCs) is a common tool used to evaluate immune system changes in clinical studies. The development of methods that would allow preservation of samples for flow cytometric analysis is important for the extension of this technology to field testing in settings where the equipment might be not readily accessible. METHODS: Three-color flow cytometric analysis was used to determine percentages of T cells and their subsets (CD3(+), CD4(+), CD8(+)), B cells (CD19(+)), and natural killer cells (CD16(+)/56(+)) in WB and PBMCs using variations of a standard stain/fix WB staining procedure (Optilyse) that included staining following fixation and freezing of fixed samples before or after staining. RESULTS: Comparable lymphocyte subset percentages in WB or PBMCs were observed regardless of Optliyse method used (all Ps >/= 0.8). However, differences in fluorescence intensity for several markers were observed across procedures. Compared with the standard stain/fix procedures, fix/stain decreased the mean fluorescence intensities for CD4, CD8, CD19 and CD16/56 in WB and PBMCs (P </= 0.03 for these markers P = 0.105 for CD8 in PBMCs). Further decreases in mean fluorescence intensity were seen with the fix/stain/freeze procedure. The stain/fix/freeze yielded intensities largely comparable to those seen with standard stain/fix procedure (P >/= 0.13), suggesting that, when the markers of interest are known at the time of field collection, implementation of this procedure might be desirable. Fix/freeze/stain resulted in diminution of intensity in general, but they tended to be more modest than those seen for fix/stain/freeze and therefore might be applicable to field studies in instances when the specific markers of interest cannot be defined upfront. CONCLUSIONS: Freezing of fixed WB and PBMCs before or after cell surface staining is a reliable method for preserving specimens in field sites for later determination of lymphocyte subset percentages, which are commonly assessed in immunodeficient and cancer patients.
Blood Preservation↗
Comparison of the Ruthenium hexammine trichloride method to other methods of chemical fixation for preservation of avian physeal cartilage.
Several methods of chemical fixation of avian physeal cartilage were compared. The Ruthenium hexammine trichloride method was compared to isotonic glutaraldehyde and neutral buffered formalin for light microscopy and paraffin embedment, and to two osmium-ferrocyanide methods and a combination of 1% glutaraldehyde and 4% formaldehyde for electron microscopy. Only the Ruthenium hexammine trichloride method prevented the loss of matrix proteoglycans and shrinkage of chondrocytes. In undecalcified paraffin-embedded cartilage, preservation of matrix and cellular detail was excellent, but Ruthenium hexammine trichloride interfered with Haematoxylin and Eosin staining. Glutaraldehyde gave more intense eosinophilia than neutral buffered formalin. Ultrastructurally, the Ruthenium hexammine trichloride method was the most consistent and gave the best overall fixation. Matrix elements and cellular and nuclear membranes were well preserved. It did result in vacuolation of the cytoplasm and mitochondria, and it increased granularity of the cytoplasm, chromatin, and rough endoplasmic reticulum. Other fixatives produced minimal vacuolation and finer granularity, but preservation was less consistent, cell/matrix contrast was often excessive, and they caused shrinkage of all chondrocytes. Large dilatations of the rough endoplasmic reticulum that appear to be cytoplasmic inclusions by light microscopy are described for the first time in avian cartilage.
Animals↗
Vitreous morphology after carbon dioxide laser irradiation.
The immediate vitreous changes produced by a continuous-wave carbon-dioxide laser beam in freshly enucleated bovine and pig eyes were studied by gross examination with a dissecting microscope, and histopathologically by light microscopy and transmission electron microscopy. The lesions produced by the laser beam on the vitreous in an air tissue interface technique were remarkable for their ring-shaped vitreous condensations. The diameter and density of these condensations increased with irradiance. With the same irradiation parameters the lesions at the anterior hyaloid membranes showed greater size and density than those in the central area of the vitreous. These vitreous condensations were demonstrated to consist of heat-denatured collagen fibrils. For light microscopic evaluation of the vitreous a modified method of tissue processing according to Szent Györgi has proven optimal in reducing vitreous shrinkage from dehydration. This method of fixation and tissue processing appeared to be useful in evaluating lesions of the vitreous and their relationship to their surroundings.
Animals↗
Determination of calcaneal ultrasound properties ex situ: reproducibility, effects of storage, formalin fixation, maceration, and changes in anatomic measurement site.
The objective of this study was to determine the reproducibility of ultrasonic bone properties with a system for measuring calcanei ex situ; the influence of changes of the measurement site; and the effects of fixation, storage, and maceration. We examined 14 fixed calcanei and 12 fresh bones. Ultrasonic measurements were performed ex situ after degassing, using an Achilles+ system and a special positioning device. The instrument precision was 0.16% for speed of sound (SOS), 1.4% for broadband ultrasonic attenuation (BUA), and 1. 8% for the stiffness index (SI). The short-term precision was 0.54%, 1.9%, and 2.8%, respectively. A defined shift of the measurement site (5 mm distal of the middle) led to unpredictable changes in ultrasound (US) properties (r = 0.65 for SOS, 0.82 for BUA, and 0.75 for SI). Embalment with 4% formalin/96% alcohol caused a systematic decrease in SOS, an increase in BUA, and a decrease in SI (mean = -12.7 units; P < 0.001), the effect increasing with time. However, values at 6 months of fixation and later were highly correlated with those in fresh specimens (r = 0.95 for the SI). Two weeks storage in degassed and normal solution had only modest effects on ultrasound properties. Maceration did not lead to a systematic increase or decrease of ultrasound variables, but introduced unpredictable changes (r = 0.64-0.94). We conclude that in comparative biomechanical studies it is feasible to measure calcaneal specimens embalmed in formalin/alcohol ex situ, if the primary interest is not in the absolute values but in the correlation with mechanical failure loads at other skeletal sites.
Aged↗
An innovative fixative for cytoskeletal components allows high resolution in colocalization studies using immunofluorescence techniques.
Using a new fixation solution, CytoSkelFix, it is now possible to obtain superior fixation and thus resolution of cytoskeletal components using immunofluorescence and fluorescence microscopy. This fixative combines rapid cell penetration and cellular crosslinking of proteins such that both preservation and resolution of cellular proteins can be detected. The cytoskeleton has proven very difficult to preserve, partly because of the lability of one of the filament systems (microtubules), and one single fixative is incapable of properly preserving microtubules, microfilaments, and intermediate filaments for localization in the same cell. Further, the motor proteins associated with the cytoskeletal elements are even more difficult to preserve, particularly simultaneously with the fiber system with which they associate. We present evidence that CytoSkelFix is a superior preservative and would be useful in fixation for all types of immunofluorescence colocalization studies where superior preservation is required.
Actins↗
Fine localization of low and high calcium dependent ATPase activities in the rat sciatic nerve.
We tried to demonstrate the electron microscopic histochemical localization of membrane Ca(2+)-ATPase activity in glutaraldehyde-fixed rat sciatic nerves. Although conventional glutaraldehyde fixatives containing impurities interfered with the reactivity of Ca(2+)-ATPase, this activity was successfully preserved in the tissues fixed with pure glutaraldehyde as well as in those fixed with paraformaldehyde. In unmyelinated nerve fibers, an ATPase activity depending on 10 mM CaCl2 was detected on the whole external surface of Schwann cell plasma membranes. In myelinated fibers, this activity was localized on the surface of Schwann cell outer loops at the paranodal region of Ranvier nodes and on the axonal membrane at the nodal region. Another activity depending on 0.1 mM CaCl2 was demonstrated on the axolemma of unmyelinated fibers. These results indicated that there may be two types of Ca(2+)-ATPase activities showing high and low affinity to calcium ions localized in peripheral nerve systems in a different manner between myelinated and unmyelinated fibers.
Animals↗
Transfer of scrapie prion infectivity by cell contact in culture.
BACKGROUND: When a cell is infected with scrapie prions, newly synthesized molecules of the prion protein PrP(C) are expressed at the cell surface and may subsequently be converted to the abnormal form PrP(Sc). In an experimental scrapie infection of an animal, the initial innoculum of PrP(Sc) is cleared relatively rapidly, and the subsequent propagation of the infection depends on the ability of infected cells to convert uninfected target cells to stable production of PrP(Sc). The mechanism of such cell-based infection is not understood. RESULTS: We have established a system in dissociated cell culture in which scrapie-infected mouse SMB cells are able to stably convert genetically marked target cells by coculture. After coculture and rigorous removal of SMB cells, the target cells express PrP(Sc) and also incorporate [35S]methionine into PrP(Sc). The extent of conversion was sensitive to the ratio of the two cell types, and conversion by live SMB required 2500-fold less PrP(Sc) than conversion by a cell-free prion preparation. The conversion activity of SMB cells is not detectable in conditioned medium and apparently depends on close proximity or contact, as evidenced by culturing the SMB and target cells on neighboring but separate surfaces. SMB cells were killed by fixation in aldehydes, followed by washing, and were found to retain significant activity at conversion of target cells. CONCLUSIONS: Cell-mediated infection of target cells in this culture system is effective and requires significantly less PrP(Sc) than infection by a prion preparation. Several lines of evidence indicate that it depends on cell contact, in particular, the activity of aldehyde-fixed infected cells.
Animals↗
Cellular fixation. A study of CytoRich Red and Cytospin Collection Fluid.
OBJECTIVE: To test a new fixative system (Roche Image Analysis System, Inc. [RIAS]) that may create a background free of blood and blood products, and to determine if the CytoRich Red system has a potential for automation of nongynecologic cytology. We compared the amount of red blood cells, background material and diagnostic ability in 40 bloody specimens prepared using the CytoRich Red system and our routine fixative, Cytospin Collection Fluid (Shandon, Inc.). STUDY DESIGN: Thirty bloody, nongynecologic specimens and 10 fresh FNA surgical specimens were prepared by adding specimen to equal volumes (10 mL) of CytoRich Red Fixative and Cytospin Collection Fluid. After initial centrifugation, the specimens were resuspended and cytocentrifuged using the Cytospin III (Shandon, Inc.) and the Hettich Universal cytocentrifuge. To test system dependency, specimens from each fixative were prepared using the alternate method of cytocentrifugation. Slides from both fixatives were stained, coverslipped and reviewed for the presence of red blood cells, background and diagnostic ability. RESULTS: Of the 40 CytoRich Red specimens, 92.5% (37) had little or no red blood cells as compared to 22.5% (9) of the 40 Cytospin Collection Fluid specimens. Seventy five percent (30) of the 40 CytoRich Red specimens showed reduction of background material in contrast to 15% (6) of the 40 Cytospin Collection Fluid specimens. Diagnostic ability using CytoRich Red was enhanced by the reduction of red blood cells and background material. CytoRich Red performed equally as well with each cytocentrifugation device. CONCLUSION: CytoRich Red reduces red blood cells and background. Nuclear and cytoplasmic stain appear improved. This allows better evaluation of the cytologic features and interpretation of bloody specimens. It is non-system dependent and can be used with any method of preparation. Also, the reduction of background and blood lends itself to adaptation in the automation of nongynecologic cytology.
Biopsy, Needle↗