A study of the ultrastructural organization of cytochromec-phospholipid membranes as revealed by various experimental treatments

Summary Two different types of cytochromec-phospholipid model membranes have been investigated by electron-microscopy. The preservation of the lamellar structure of the cytochromec plus phospholipid-water system after extraction of 88% phospholipids with aqueous acetone followed by only aldehyde fixation is revealed.Pronase digestion of this type of model membrane leads to removal of 80% of the protein and loss of ordered structures as revealed by aldehyde post-fixation.It is shown that the hydrated isooctane-soluble cytochromec-phospholipid complex retained the lamellar structures after extraction of 75% phospholipids followed by separate aldehyde and osmium tetroxide fixations. Pronase treatment of this type of model membrane leads to removal of 25 to 30% of the protein. After aldehyde fixation of these pronase-treated samples the triple-layered structures are preserved.The structural integrity of both types of model membranes after organic extractions with aldehyde pre- or post-fixation is thought to be the result of cross-linkage of the protein by aldehyde polymers on both surfaces and through lipid bilayers. Possible localization of cytochromec in investigated model membranes is discussed.     

Parathyroid ultrastructure after aldehyde fixation, high-pressure freezing, or microwave irradiation

Parathyroid cell variants, commonly observed in parathyroid glands fixed by immersion in glutaraldehyde, are believed to be the result of cyclic changes in the course of parathyroid hormone secretion. Immersion of bovine parathyroid glands in a mixture consisting of 1% glutaraldehyde, 1.5% formaldehyde, and 2.5% acrolein, followed by post-fixation in 1% osmium tetroxide, resulted in high uniformity with only one cell variant, whereas the same fixation procedure led to disruption of cell membranes and formation of cell variants in rat parathyroids. Parathyroid glands of both cattle and rats prepared by high-pressure quick-freezing and subsequent freeze-substitution contained only one cell variant. Excellent preservation of the ultrastructure of bovine and rat parathyroids, also exhibiting only one cell variant, was achieved by microwave irradiation in the presence of 2.5% glutaraldehyde in Na-cacodylate followed by post-fixation with OsO4 in Na-cacodylate or s-collidine, both containing Ca2+ and Mg2+. Use of the appropriate buffer, as well as osmication, is essential for successful fixation utilizing microwave energy. The main effects are considered to be heating specimens within sufficient short periods and enhancement of subsequent osmium fixation. The results support the idea, arising after examination of perfusion-fixed parathyroid tissue, that parathyroid cell variants occur during improper aldehyde fixation rather than that they express functional diversity.     

Preservation of cartilage matrix proteoglycans using cationic dyes chemically related to ruthenium hexaammine trichloride.

We tested various cationic dyes chemically related to ruthenium hexaammine trichloride (RHT) [i.e., the RHT-cyclohexanedione complex (RHT-CC), pentaamine ruthenium N-dimethylphenylenediimine trichloride (PRT), tris-(bipyridyl)ruthenium (II) chloride (TRC), tris (bipyridyl) iron (II) chloride (TIC), and cobalt hexaammine trichloride (CHT)] for their effectiveness in precipitating cartilage matrix proteoglycans in situ. Dyes were introduced into media at the onset of processing and were present throughout both aldehyde fixation and osmium tetroxide post-fixation. Contrary to expectation, most of the dye-proteoglycan complexes generated and stable under aldehyde fixation conditions were found to be unstable during post-fixation despite the continuing presence of the dye. A similar phenomenon was also found for the cationic dyes commonly used for precipitation of proteoglycans in cartilage tissue sections (such as Acridine Orange, Alcian Blue, Azure A, Methylene Blue, and Ruthenium Red). Only two dyes, i.e., RHT and the newly tested RHT-CC, formed proteoglycan precipitates sufficiently stable to resist disruption and extraction during osmium tetroxide post-fixation. The latter may be particularly useful in semiquantitative analyses of proteoglycan content in unstained tissue sections owing to its intense brown-black color. For applications in which the osmium tetroxide post-fixation step may be omitted, TRC and PRT may also be valuable for semiquantitative histochemistry by virtue of their stable fluorescence and intense violet color signals, respectively.     

Plasmatubules: fact or artefact?

Plasmatubules are tubular evaginations of the plasmalemma associated with sites where high solute flux occurs between apoplast and symplast. Plasmatubules of the scutellar epithelial cells of germinating barley (Hordeum vulgare L.) have been examined following a variety of fixation methods. Of the aqueous fixations, primary aldehyde fixation with osmium post-fixation and osmium as the primary fixative gave comparable images, whilst potassium permanganate resulted in some distortion of the tissue in general including dilation of the tubular evaginations of the plasmalemma. Freeze-fixation and substitution with acetone and acetone-osmium gave images of the plasmalemma comparable to those obtained by the aqueous aldehyde and osmium methods. The similarity of structure with aldehyde or osmium and freezing as the primary fixation is taken to indicate that plasmatubules are real and not artefacts resulting from the fixation procedure.     

Effects of lung volume, bronchoconstriction, and cigarette smoke on morphometric airway dimensions

To examine the role of airway wall thickening in the bronchial hyperresponsiveness observed after exposure to cigarette smoke, we compared the airway dimensions of guinea pigs exposed to smoke (n = 7) or air (n = 7). After exposure the animals were anesthetized with urethan, pulmonary resistance was measured, and the lungs were removed, distended with Formalin, and fixed near functional residual capacity. The effects of lung inflation and bronchoconstriction on airway dimensions were studied separately by distending and fixing lungs with Formalin at total lung capacity (TLC) (n = 3), 50% TLC (n = 3), and 25% TLC (n = 3) or near residual volume after bronchoconstriction (n = 3). On transverse sections of extraparenchymal and intraparenchymal airways the following dimensions were measured: the internal area (Ai) and internal perimeter (Pi), defined by the epithelium, and the external area (Ae) and external perimeter (Pe), defined by the outer border of smooth muscle. Airway wall area (WA) was then calculated, WA = Ae - Ai. Ai, Pe, and Ae decreased with decreasing lung volume and after bronchoconstriction. However, WA and Pi did not change significantly with lung volume or after bronchoconstriction. After cigarette smoke exposure airway resistance was increased (P less than 0.05); however, there was no difference in WA between the smoke- and air-exposed groups when the airways were matched by Pi. We conclude that Pi and WA are constant despite changes in lung volume and smooth muscle tone and that airway hyperresponsiveness induced by cigarette smoke is not mediated by increased airway wall thickness.     

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.     

Antigen preservation tests for immunocytochemical detection of cytoskeletal proteins: influence of aldehyde fixatives

The effects of aldehyde fixatives on immunochemical detection of cytoskeletal proteins were demonstrated by applying several quantitative assays to evaluate antigen conservation. Immunologically detectable brain spectrin (240/235) was measured by dot-immunobinding and quantitative immunodot assay using a polyclonal antibody. Paraformaldehyde fixation led to a 43-66% reduction in brain spectrin (240/235) immunodetection, and increasing glutaraldehyde concentrations decreased the immunological detection even more. Quantitative cryosection immunoassay and immunocytochemical localization confirmed the aldehyde sensitivity of brain spectrin (240/235). Brain spectrin (240/235) immunoreactivity decreased with increasing protein crosslinking and was dependent on glutaraldehyde concentration and post-fixation period. The assays were also used to test for conservation of antigenicity of neurofilament proteins by two monoclonal antibodies. Neurofilament detection was abolished in brain tissue after aldehyde fixation. The described methods allow screening within 24 hr of many fixation conditions by use of purified proteins as well as brain tissue samples, and allow an estimate of fixative influence on the conservation of protein antigenicity.     

Enhanced ultrastructural preservation of cartilage proteoglycans in the extended state

We investigated the preservation of proteoglycan (PG) structure in rat epiphyseal cartilage using N-N-dimethylformamide (DMF) dehydration before embedding. After aldehyde fixation, specimens with and without routine osmium post-fixation were dehydrated in graded DMF and embedded in either Spurr's resin or Lowicryl K4M resin. Standard ethanol dehydration with Spurr or Lowicryl embedding techniques resulted in the formation of condensed PGs, called matrix granules. DMF dehydration before embedding greatly improved the preservation of PG structure and resulted in an extended appearance of PGs closely resembling the fine filamentous network of cartilage tissues processed by rapid freezing and freeze-substitution. However, en bloc staining of aldehyde-fixed specimens with cationic reagents before or during DMF dehydration induced the condensation of PGs and resulted in the formation of matrix granules. These observations demonstrate that DMF, a mild dehydration agent, dramatically improves PG preservation without a harmful effect on aldehyde-fixed PG structure and can be utilized regardless of routine post-fixation.     

Desialylation of core type 1 O-glycan in the equine embryonic capsule coincides with immobilization of the conceptus in the uterus

During the second and third weeks of pregnancy, the equine conceptus expands rapidly while it is enclosed within a glycan capsule. Around day 16 of gestation, the conceptus loses its mobility in the uterus by a process termed 'fixation', coinciding with various changes in the capsule. Here, we compared the structure of the carbohydrate moieties expressed by the capsule during pre- and post-fixation periods. The glycan structures were studied by chemical analyses in combination with mass spectrometry. Capsule material from conceptuses collected before fixation (days 13-16) was observed to carry a sialylated core type 1 O-linked glycan, Neu5Ac-(2-->3)-Gal-(1-->3)-GalNAc-(1-->Ser/Thr. By comparison, analysis of post-fixation capsules (days 17-19) revealed a desialylated core type 1, Gal-(1-->3)-GalNAc-(1-->Ser/Thr. The equine embryonic capsule also furnished 4-substituted GlcNAc, 4-substituted Glc and 2,3,4,6-tetrasubstituted Glc residues, the concentrations of which did not change between pre- and post-fixation stages. The loss of sialic acid from the sialylated core type 1 in the capsule appears to be directly related to successful fixation of the conceptus, and thus critical to the continuance of pregnancy in horses.     

Effect of Pre-Fixation Delay and Freezing on Mink Testicular Endpoints for Environmental Research

There is growing interest in using wild animals to monitor the real-life cocktail effect of environmental chemicals on male reproduction. However, practical difficulties, such as long distances to the laboratory, generally prolong the time between euthanisation and specimen handling. For instance, tissue fixation is often performed on frozen material or on material where deterioration has started, which may affect tissue morphology. This study examined the effect of pre-fixation delay and freezing on mink testicular endpoints in order to determine robust endpoints in suboptimally handled specimens. Sexually mature farmed mink (n=30) selected at culling were divided into six groups and subjected to different time intervals between euthanisation and fixation or freezing: 0 hours (fixed immediately post mortem), 6 hours, 18 hours, 30 hours, 42 hours, or frozen 6 hours post mortem and thawed overnight. Unaffected endpoints when pre-fixation storage was extended to 30 hours included: area and diameter of the seminiferous tubules, length and weight of the testes, and acrosomes marked with Gata-4. Epithelial height, Sertoli cells marked with Gata-4 and cell morphology were affected endpoints after 6 hours of storage. Freezing the tissue prior to fixation severely altered cell morphology and reduced testicular weight, tubular diameter and area. Morphological changes seen after 6 hours included shredded germ cells and excess cytoplasm in seminiferous tubular lumen, chromatin rearrangements and increased germ cell death. Extended delay before fixation and freezing affected many endpoints in the mink testicular tissue. Some of these endpoints may mimic chemically induced effects, which is important to consider when evaluating specimens from wild animals for environmental toxicity.     

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18-21 particles/1000 nm of LRE) with fairly regular interspacing (45-65 nm) as reported previously.(ABSTRACT TRUNCATED AT 250 WORDS)     

Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins

Summary In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO₄ post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.     

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

Summary The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18–21 particles/1000 nm of LRE) with fairly regular interspacing (45–65 nm) as reported previously.This is the first report to identify the three-dimensional ultrastructure of anionic sites of GBM, and provides new information on the location and distribution of anionic sites in the glomerular capillary wall. In addition, these studies suggest that several chemical procedures used in conventional transmission electron microscopy to visualize PEI tracers, may produce structural changes and disarrangement of PEI particles that can be avoided with the quick-freezing and deep-etching method.     

Short-term load bearing capacity of osteochondral autografts implanted by the mosaicplasty technique: an in vitro porcine model

Articular surface congruency and graft stability are considered essential factors in the success of osteochondral grafting; however, quantitative measures of short-term load bearing capacity of grafts implanted by the mosaicplasty technique have not been reported. The purpose of this study was to develop a live tissue in vitro model to examine short-term fixation strength of mosaicplasty autografts immediately after and 1 week following graft implantation. Cylindrical osteochondral autografts were implanted in vitro by the mosaicplasty technique on five pairs of porcine femoral condyles within one and a half hours of animal sacrifice. Immediately following the surgical procedure, graft push-in and pull-out strength tests as well as indentation tests to determine modulus of the surrounding cancellous bone were performed on half of the specimens from the distal femurs of each animal. The remaining specimens, matched for location in the contralateral leg, were incubated in culture medium for 7 days prior to performing the same set of mechanical tests. Averaged push-in and pull-out graft fixation strength decreased 44% from 135.7 to 75.5N over the 7-day period, while no change in modulus was detected in the surrounding cancellous bone. These in vitro results demonstrate a substantial deterioration of short-term fixation strength of mosaicplasty grafts from the immediate post-operative state. Such a reduction in short-term graft load bearing capacity may pose a threat to the surgically established articular surface congruency and blood vessels formed during the early stages of the healing response.     

Adaptation of the left ventricle to exercise-induced hypertrophy

Cardiac functional and structural adaptations to exercise-induced hypertrophy were studied in 68 pigs. Pigs were exercise trained on a treadmill for 10 wk. Sequential measurements were made of cardiac dimensions, [left ventricular end-diastolic diameter (EDD), changes in diameter (delta D%), wall thickness (WTh), wall thickening (WTh%), left ventricular pressure (LVP), time derivative of pressure (dP/dt), stroke volume, total body O2 consumption (VO2), blood gases, and systemic hemodynamics] at rest and during moderate and severe exercise. Postmortem studies included morphometric measurements of capillary density, arteriolar density, mitochondria, and myofibrils. All of the exercise-trained pigs showed significant increases in aerobic capacity. Maximum O2 consumption (VO2 max) increased by 37.5% in group 1 (moderate exercise training) and 34% in group 3 (heavy exercise training). Cardiac hypertrophy ranged from less than 15% in a group (n = 8) subjected to moderate exercise training to greater than 30% in a group (n = 11) subjected to heavy exercise training. Before training, exercise was characterized by a decreasing EDD during progressive exercise; this was reversed after exercise training. Stroke volume and end-diastolic volumes during exercise showed a highly significant increase after exercise training and hypertrophy. Morphometric measurements showed that mitochondria and cell membranes increased with increasing myocyte growth in all exercise groups, but there was only a partially compensated adaptation of capillary proliferation. Arteriolar number and length increased in all exercise groups. Intrinsic contractility as measured by delta D%, WTh%, or left ventricular dP/dt did not increase with exercise training and in some instances decreased. Therefore, left ventricular adaptation to strenuous exercise in the pig heart is primarily one of changes in left ventricular dimensions and a compensated hypertrophy. Exercise-induced increases in EDD and stroke volume can be accounted for by decreases in peripheral resistance and increased cardiac dimensions.     

THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.     

Surface:volume relationship in cardiac myocytes studied with confocal microscopy and membrane capacitance measurements: species-dependence and developmental effects.

The quantitative analysis of the contribution of ion fluxes through membrane channels to changes of intracellular ion concentrations would benefit from the exact knowledge of the cell volume. It would allow direct correlation of ionic current measurements with simultaneous measurements of ion concentrations in individual cells. Because of various limitations of conventional light microscopy a simple method for accurate cell volume determination is lacking. We have combined the optical sectioning capabilities of fluorescence laser scanning confocal microscopy and the whole-cell patch-clamp technique to study the correlation between cell volume and membrane capacitance. Single cardiac myocytes loaded with the fluorescent dye calcein were optically sectioned to produce a series of confocal images. The volume of cardiac myocytes of three different mammalian species was determined by three-dimensional volume rendering of the confocal images. The calculated cell volumes were 30.4 +/- 7.3 pl (mean +/- SD) in rabbits (n = 28), 30.9 +/- 9.0 pl in ferrets (n = 23), and 34.4 +/- 7.0 pl in rats (n = 21), respectively. There was a positive linear correlation between membrane capacitance and cell volume in each animal species. The capacitance-volume ratios were significantly different among species (4.58 +/- 0.45 pF/pl in rabbit, 5.39 +/- 0.57 pF/pl in ferret, and 8.44 +/- 1.35 pF/pl in rat). Furthermore, the capacitance-volume ratio was dependent on the developmental stage (8.88 +/- 1.14 pF/pl in 6-month-old rats versus 6.76 +/- 0.62 pF/pl in 3-month-old rats). The data suggest that the ratio of surface area:volume of cardiac myocytes undergoes significant developmental changes and differs among mammalian species. We further established that the easily measurable parameters of cell membrane capacitance or the product of cell length and width provide reliable but species-dependent estimates for the volume of individual cells.     

Ultrastructural observations on tissues processed by a quick-freezing,rapid-drying method: Comparison with conventional specimen preparation

A quick-freeze, rapid-dry method for processing unfixed tissue for electron microscopy has been developed. The technique employs freezing on a cryogenchilled metal surface and drying in a cryosorption vacuum apparatus that allows osmium-vapor fixation and epoxy-resin embedment under high vacuum. Liver, kidney, bone marrow, and monolayer cultures of ventricular myocytes were selected as tissue specimens representing a wide range of physical properties, to demonstrate the practical aspects of achieving good ultrastructural morphology by freeze drying. A comparison was made between freeze drying and conventional processing using aldehyde fixation and alcohol dehydration. The preservation of cellular ultrastructure achieved by freeze drying allowed the identification of specific cell types within each specimen. Membranous organelles were well preserved, surrounded by cytoplasmic ground substance devoid of ice crystal damage. Electron-dense material was observed within the rough endoplasmic reticulum and Golgi cisternae and vesicles of frozen-dried, but not conventionally processed cells. This suggests the preservation by freeze drying of cytoplasmic components otherwise extracted from the cell by solvent exposure.     

Semiquantitative assessment of c-erbB-2 (HER-2) status in cytology specimens and tissue sections from breast carcinoma

OBJECTIVE: To determine whether various methods of fixation of surgical pathology specimens from breast carcinomas would influence the outcome of evaluation of the expression levels of c-erbB-2 (HER-2). For this, comparisons were made between (1) alcohol-fixed (95%) and air-dried smears from fresh surgical pathology specimens of breast carcinomas, and (2) formalin-fixed, paraffin-embedded tissue sections of the same specimens. STUDY DESIGN: Alcohol-fixed and air-dried smears or touch preparations were made from 30 fresh mastectomy/lumpectomy surgical pathology specimens from breast carcinomas. Immunohistochemistry was performed using the c-erbB-2 primary antibody against the extracellular domain of the c-erbB-2 gene product. Staining was simultaneously performed on formalin-fixed, paraffin-embedded tissue sections of the same specimens. A semiquantitative approach was used for evaluation of immunostaining by three independent investigators, and a consensus was reached. RESULTS: A total of 30 cases were reviewed. Tissue positivity was determined for c-erbB-2 in: 73% of alcohol-fixed specimens (n = 13 [3+] and n = 9 [2+]), 67% of air-dried smears (n = 9 [+3] and n = 11 [+2]) and 47% (n = 8 [+3]) and n = 6 [+2]) of formalin-fixed, paraffin-embedded tissue specimens. All formalin-fixed tissue specimens that were determined to positively express c-erbB-2 were also found to be positive on the alcohol-fixed smears. CONCLUSION: The incidence of c-erbB-2 expression in fresh cytologic material is significantly higher (P < .05) than in formalin-fixed, paraffin-embedded tissue. Alcohol-fixed smears demonstrate a slightly higher percentage of cell staining and stronger intensity of c-erbB-2 expression than the matched, air-dried smears. This is a sensitive and simple processing method that can be routinely applied in surgical pathology or fine needle aspiration biopsy specimens for the detection of c-erbB-2 (HER-2), with clinical implications.     

Shape and Volume Changes in Frog Erythrocytes Following Ultraviolet Radiation

Frog erythrocytes in Ringer's solution were exposed to ultraviolet radiation and then followed in camera lucida drawings for changes in shape and dimension. Cell thickness was found to increase while cell width remained constant throughout the period prior to hemolysis. The cell shortened and bulged at the ends during the middle third of the prolytic period while a region around the cell center remained constricted. When this constricted region gave way, the cell became spherical and hemolyzed. Cell volume as calculated from the cell's dimensions increased linearly with time throughout the prolytic period to hemolysis then dropped rapidly to a constant value somewhat higher than the original cell volume. These changes in shape and volume are consistent with a colloid osmotic type of hemolysis but with other factors acting to limit the rate of swelling and the forms assumed during the swelling process. The relationship between the time of hemolysis and the cell surface area exposed to the ultraviolet is discussed as it applies to the site of ultraviolet damage.