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PubMed1999

The bone marrow plasma cell labeling index by flow cytometry.

The bone marrow plasma cell labeling index is the most important prognostic indicator for patients with multiple myeloma. Traditionally, this test has been performed as a two color immunofluorescent microscope technique which is time consuming and requires a degree of subjectivity in its interpretation. We have assessed various adaptations of this method to flow cytometry. A bromodeoxyuridine method has been compared with a propidium iodide DNA method to detect cells in S phase and CD38-FITC has been compared with CD38-FITC + CD138-FITC and CD38-biotin + streptavidin FITC to identify plasma cells. The mean channel fluorescent intensity of the plasma cell peaks for each of these markers was 12. 7, 17.4 and 35.3 respectively demonstrating the superiority of CD38-biotin + streptavidin FITC. Analysis after propidium iodide staining provided a good correlation with the slide technique (r = 0. 71; P < 0.0001) but the bromodeoxyuridine method did not correlate with the slide method (r = 0.09; P = NS). The labeling index values obtained from either of the flow methods were greater than the microscopic method. Thus a labeling index of >4% will replace the traditional >1% threshold for identifying patients with a significantly increased labeling index. The advantages of the new method are that it takes less time to perform, is more objective and provides additional data on ploidy and cell cycle status.

ADP-ribosyl Cyclase↗
PubMed1998

Sustained neonatal hyperthyroidism in the rat affects myelination in the central nervous system.

We have carried out a study of the effects of sustained neonatal hyperthyroidism on myelin and on the oligodendroglial cells, in an effort to obtain further insight into the molecular mechanisms underlying the action of thyroid hormones on the central nervous system (CNS). Expression of the mRNAs of myelin basic protein (MBP) myelin proteolipid protein (PLP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), transferrin, and c-Jun was investigated in 10- and 17-day-old normal and hyperthyroid rats, using Northern blot analysis. At 10 days of age, the levels of all the explored mRNAs were markedly higher in the experimental animals. The mRNA of transferrin showed a ninefold increase over control values, suggesting the possibility that this putative trophic factor might act as one of the mediators in the action of thyroid hormones. At 17 days of age on the other hand, the levels of all the mRNAs decreased markedly, reaching values below control, except for c-Jun, which remained higher than in normals. At 70 days of age, hyperthyroid rats showed clear evidence of myelin deficit, in agreement with previous results of our laboratories (Pasquini et al.: J Neurochem 57: Suppl S124, 1991). Immunocytochemistry of 70-day-old rat brain tissue sections showed a substantial reduction in the amount of MBP-reacting structures and a marked decrease in the number of oligodendroglial cells. Although the above-mentioned results could be the consequence, as proposed by Barres et al. (Development 120:1097-1108, 1994) and Baas et al. (Glia 19:324-332, 1997) of a premature arrest in oligodendroglial cell proliferation followed by early differentiation, the persistent high levels of expression of c-Jun, together with the dramatic decrease in the number of oligodendrocytes, suggested the possibility that prolonged hyperthyroidism could activate apoptotic mechanisms in the myelin forming cells. Using propidium iodide-labeled isolated oligodendroglial cells, we found, by flow cytometry, a significant increase in the number of apoptotic/hypo-diploid propidium iodide-positive cells. These results indicate that one of the actions of sustained levels of thyroid hormones in the neonate rat is to increase oligodendroglial cell death by apoptosis.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1989

Ethanol fixation of bladder irrigation specimens for flow cytometric analysis. A multiinstitutional study from the bladder cancer flow cytometry network.

Ethanol preservation of voided and catheterized urine has long been the standard for urinary cytologic study. In this report ethanol preservation of bladder irrigation specimens was evaluated for flow cytometric analysis in a multiinstitutional study requiring specimen transport. Specimens from ten patients obtained at one center were preserved for varying periods of time in 50% ethanol, then distributed by express mail to four other participating centers, up to 3000 miles distant. On receipt, from 1 to 3 weeks after collection, the samples were processed and examined by flow cytometric study using propidium iodide as the fluorochrome. Forty-six of the 50 (92%) analyzed specimens gave satisfactory histograms. In 41 of the 46 adequate samples (89%), an aneuploid peak in the alcohol preserved (propidium iodide stained) specimen correlated well with the fresh (acridine orange stained) specimen. However, there was variable loss of DNA stainability with a broadening of the coefficient of variation in some alcohol-preserved specimens and an increase in cellular debris so that measurements of DNA index and percent hyperdiploid cells were considered unreliable. The authors conclude that ethanol preservation of bladder irrigation specimens for short periods of time may be a feasible alternative when flow cytometric analysis cannot be carried out on fresh specimens, but that this is not optimal fixation for specimens that must be transported and further studies of other fixatives are recommended.

Aneuploidy↗
PubMed2003

Serum amyloid P component binds to late apoptotic cells and mediates their uptake by monocyte-derived macrophages.

OBJECTIVE: Some pentraxins, such as C-reactive protein, bind to apoptotic cells and are involved in the clearance of these cells. We undertook this study to determine whether serum amyloid P component (SAP; a pentraxin that, when deficient in mice, results in lupus-like disease) binds to apoptotic cells and to assess the functional consequences of SAP binding for their phagocytosis by macrophages. METHODS: Human peripheral blood monocytes were isolated and cultured for 7 days to obtain monocyte-derived macrophages. Jurkat cells were irradiated with ultraviolet B to induce apoptosis. After 4 hours, a mean +/- SEM of 54.0 +/- 5.1% of these cells stained with annexin V and were propidium iodide negative (early apoptotic [EA] cells). After 24 hours, 77.3 +/- 2.7% of cells stained positive with both annexin V and propidium iodide (late apoptotic [LA] cells or secondary necrotic cells). EA and LA cells were incubated with fluorescein isothiocyanate-labeled SAP in the presence or absence of Ca(2+), and binding was measured by flow cytometry. Phagocytosis was tested by incubation of macrophages with EA or LA cells in the presence of normal human serum (NHS) and quantified as a phagocytosis index (PI; number of Jurkat cells internalized by 100 macrophages). Experiments were repeated with SAP-depleted serum and after reconstitution with increasing concentrations of SAP. RESULTS: The majority of LA cells did bind SAP in the presence of Ca(2+), whereas EA cells did not. SAP depletion of NHS resulted in a 50% decrease in the PI for LA cells, and complete restoration of the PI could be demonstrated with SAP reconstitution up to 100 microg/ml. SAP depletion had no effect on phagocytosis of EA cells. CONCLUSION: SAP binds to LA cells and is involved in the phagocytosis of these cells by human monocyte-derived macrophages. This may have consequences for diseases such as systemic lupus erythematosus, in which phagocytosis of apoptotic cells is decreased.

Apoptosis↗
PubMed2003

A new strategy for the design of water-soluble synthetic receptors: specific recognition of DNA intercalators and diamines.

Water-soluble zinc bisporphyrin receptors 1 and 2 having two Lewis acidic sites (zinc) in the hydrophobic environment consisting of alkyl chains and a bisporphyrin framework, and covered with hydrophilic exterior (twelve or eighteen carboxyl groups) were prepared. The receptors show high affinity for diamines and DNA intercalators in water where the binding constants K(a) are of the order of 10(7) and 10(8) M(-1), respectively. Diamines and DNA intercalators are bound to the receptor through different mechanisms. Diamines are bound through hydrophobic interactions and zinc-nitrogen interactions, while DNA intercalators are bound through hydrophobic interactions and charge-transfer interactions. Flexible alkyl chains can make van der Waals contact with guests and create a hydrophobic environment around the bound guest by an induced-fit-type mechanism. For the binding of DNA intercalators, the following features are noteworthy: 1). Binding constants are similar between the zinc porphyrins and zinc-free porphyrins; 2). the binding constant is larger for the guest having the lower LUMO; this indicates the important contribution of charge-transfer interactions to binding; 3). the hydrophobic and cationic nature of DNA intercalators is substantially important, and 4). higher ionic strength reduced the binding affinities; this shows a moderate contribution of electrostatic interactions. The conformational instability of the receptors also contributes to the tight binding: hydrophobic and electrostatic interactions cannot both be favorable at the same time in the guest-free receptor. Enthalpy-entropy compensation observed for the binding of diamines and DNA intercalators is characterized by a relatively small slope (alpha=0.74) and a large intercept (beta=7.75 kcal mol(-1)) in the DeltaH degrees versus TDeltaS degrees plot; this shows that a conformational change of receptors and a significant desolvation occur upon binding. The receptor can competitively bind to propidium iodide to deprive DNA of the intercalated propidium iodide. These features of water-soluble receptors consisting of a rigid framework and flexible side chains with a large solvent-accessible area are in contrast to highly preorganized rigid receptors, and they can provide useful guidelines for rational design of induced-fit artificial receptors in water.

Binding, Competitive↗
PubMed1982

The distribution and origin of serotonin-containing fibers in the septal area: a combined immunohistochemical and fluorescent retrograde tracing study in the rat.

The distribution of 5-hydroxytryptamine (serotonin, 5-HT)-containing nerve fibers and terminals in the septal area of the rat was studied by using immunohistochemistry with specific antibodies to 5-HT in combination with fluorescent retrograde tracing methods. The 5-HT innervation of the septum is heterogeneous with regard to both the morphology of individual processes and the density of distribution in different parts of the septum. Three major classes of 5-HT like immunoreactive processes can be distinguished: 1) thin, convoluted fibers with small, round or elongated varicosities; 2) thick and relatively straight fibers with few varicosities; and 3) pericellular plexuses with large varicosities in close association with perikarya in the lateral septum. Three areas of the septum receive a prominent innervation by 5-HT processes: the diagonal band of Broca, the ventral part of the lateral septum, and an area bordering the medial edge of the islands of Calleja (insula magna). Whereas the two latter areas contain dense terminal networks, the diagonal band of Broca is occupied primarily by 5-HT fibers en route to other parts of the septum. Intraseptal injections of HRP or fluorescent dyes (granular blue, propidium iodide) resulted in retrograde labeling of neuronal cell bodies in several nuclei of the brainstem which are known to contain 5-HT neurons: the dorsal raphe, the median raphe, the nucleus reticularis tegmenti pontis, the raphe pontis, and the raphe magnus. Where fluorescent retrograde tracing was performed with 5-HT immunohistochemistry on the same tissue section, a prominent 5-HT containing pathway and a non-5-HT-containing pathway from the raphe nuclei to the septum were revealed. Finally, double retrograde fluorescent labeling after injections of granular blue or propidium iodide into the septum and entorhinal area respectively of the same rat revealed extensive branching of the raphe efferents. Thus, individual raphe neurons may simultaneously connect with septum and the entorhinal area, two structures essential for normal hippocampal function.

Animals↗
PubMed1980

SV40-infected muntjac cells: cell cycle kinetics, cell ploidy and T antigen concentration.

Muntjac cells in which the SV40 virus neither readily causes transformation nor replicates were used to study the effect of SV40 infection on cell ploidy and the influence of ploidy on the concentration of T antigen, which is required for the initiation of viral DNA synthesis. Both the DNA content, as measured by the flow microfluorometry of propidium iodide-DNA fluorescence, and the average number of chromosomes per cell indicated that infection with SV40 did not alter the ploidy of the host cell. SV40 infection had no effect on the ploidy distribution of muntjac cells. After immunofluorescence staining with anti-T serum and fluorescein-labeled anti-gamma G, infected and uninfected cultures were compared. In uninfected cells incubated with a 1:20 dilution of anti-T serum no fluorescence could be observed by fluorescence microscopy, but when examined by flow microfluorometry, fluorescence was detected after staining with as little as 1000-fold diluted antiserum. Determination of the amount of T antigen and DNA content in the same cell by simultaneous measurement of fluorescein isothiocyanate-conjugate and propidium iodide fluorescence, indicated that the cellular concentration of T antigen did not vary with the ploidy of the genome or the number of nuclei per cell. These results suggest that gene dosage is not a factor which determines the permissive environment for SV40 replication.

Animals↗
PubMed1982

Permeabilization of lymphocytes with polyethylene glycol 1000. Discrimination of permeabilized cells by flow cytometry.

The toxicity of polyethylene glycol 1000 (PEG) used similarly as in cell hybridization experiments, has been studied by flow cytometry, measuring the light scattering and fluorescence distributions of PEG-treated human lymphocytes stained with propidium iodide, fluorescein diacetate and acridine orange (AO). The sensitivity of these tests to detect permeabilized, or potentially dead cells, was equal. In addition, PEG proved to interfere with AO staining most likely through the inhibition of its binding to nucleic acids. The decrease of AO fluorescence in cells killed by PEG was unexpected since intercalation of propidium iodide was the same as in alcohol fixed cells. Permeabilization of cells by PEG appears to be an all-or-none phenomenon, accompanied by entrance of PEG into the cells. The findings are described in the context of a review of the currently used flow cytometric techniques to discriminate viable and lethally affected cells; also, the problems of interpretation are discussed.

Acridine Orange↗
PubMed1983

Optimization of mammalian chromosome suspension preparations employed in a flow cytometric analysis.

Metaphase chromosomes from Chinese hamster V-79 cells have been stained with propidium iodide and their DNA content was measured by flow cytometry. The chromosomes in suspension must be stabilized during the preparation to preserve their morphological integrity. In order to obtain high resolution in the flow karyotype, several modifications were made to the method used to prepare chromosome suspensions. The influence of several ions, detergents and detergent mixtures was investigated. Magnesium (3-5 mM) together with sodium (10 mM), Tris (10 mM) and Triton X-100 (0.4%) gave the best results. In contrast, sodium and potassium exerted no effect, while calcium had a negative effect on the preparation as measured with flow cytometry. Compared with a solution of potassium chloride + propidium iodide, chromosomes prepared in Tris buffer resolved better with less background. A histogram quality factor which contains the variables peak height, depth of valley and coefficient of variation, was used to quantitate the histograms.

Animals↗
PubMed1984

Preparation of chromosome suspensions for flow cytometry.

Certain variables in the preparation of chromosome suspensions for flow cytometric analysis have been investigated. The optimal conditions have been determined. The results of this series of experiments have been incorporated to yield a preparation protocol that gives chromosome profiles with a low amount of small particle debris and few chromosome clumps. The method reduces variability that results from sample preparation. Chromosomes are optimally isolated in a hypotonic solution buffered to pH 8.0. MgSO4 and dithiothreitol added to the buffer reduce the number of clumps and small fluorescent particles. The presence of MgSO4 also stabilizes the chromosomes and precludes the need for other stabilizing agents such as propidium iodide. When the swelling buffer developed in this investigation is used, unstained chromosomes are stable for at least 1 wk if stored at 4 degrees C. The preparation procedure can be used with the DNA stains, propidium iodide, Hoechst 33258, and mithramycin. Preliminary experiments show that this procedure can also be used for bivariate analysis of human and Chinese hamster chromosomes. The importance of this improvement for studies on chromosome damage caused by irradiation or mutagens is discussed.

Animals↗
PubMed1988

DNA flow cytometric analysis of mouse seminiferous epithelium.

In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination-assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase-contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide. DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage-specific manner. DNA histograms of stages I-VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)-binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX-XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.

Animals↗
PubMed1989

Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content.

Flow cytometry is a useful tool for measuring DNA content and differentiation as expressed by cell surface markers. We have extended this technology to measure simultaneously either surface, cytoplasmic, or nuclear antigens (particularly oncoproteins) with DNA content. Mononuclear blood cells isolated from normal subjects and HL60 leukemic cells were permeabilized and fixed in suspension utilizing 40 micrograms/ml lysolecithin and 1% paraformaldehyde. A range of lysolecithin concentrations in 1% paraformaldehyde was studied to optimize permeabilization of the antibodies to the cell interior without destroying cell integrity. The optimal concentration (40 micrograms lysolecithin/ml) resulted in good cell recovery with a high percentage of cells positive for surface and intracellular antigens. Cells are first stained with fluorescein isothiocyanate conjugated (FITC) antimyeloperoxidase (an azurophil granule enzyme), or with an anti-c-myc antibody and FITC goat anti-mouse IgG F(ab')2. Cells are then incubated with RNase and stained for DNA content with propidium iodide. Alternatively, cells were stained for the cell surface markers Leu M3, OKM1, or the transferrin receptor and were then fixed and permeabilized and stained with propidium iodide. Using this method, we correlated cytoplasmic, nuclear, or cell surface antigens with cell cycle kinetics. This technique should be useful for studies of cellular differentiation and proliferation.

Antigens, Surface↗
PubMed1992

Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry.

Mouse thymocytes readily undergo apoptosis-associated DNA degradation upon exposure to glucocorticoids or ionizing radiation. It has been previously shown that flow cytometric cell cycle analysis of propidium iodide-stained apoptotic thymocytes results in the appearance of a distinct cell cycle region (the A0 region) below the G0/G1 region. Cells in this region were shown to be undergoing apoptosis, and determination of apoptosis by flow cytometric analysis was proposed as a superior method for evaluating thymocyte apoptosis. In this study, a variety of DNA binding dyes with diverse primary binding mechanisms were evaluated for their ability to detect glucocorticoid and ionizing radiation-induced apoptosis in mouse thymocytes. Apoptotic thymocytes stained with DNA binding dyes from the phenanthridinium, acridine, actinomycin, chromomycinone, anthracycline, and bisbenzimidazole groups all demonstrated clearly defined A0 regions with percentages comparable to those obtained for propidium iodide. These results indicate that the appearance of the A0 region is not dependent on a particular dye binding characteristic and may be the consequence of extensive changes in chromatin structure resulting in a significant degree of dye exclusion.

Acridines↗
PubMed1992

Streptavidin-based quantitative staining of intracellular antigens for flow cytometric analysis.

A streptavidin-biotin-based three-step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40-50-fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two-step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two-step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 micrograms/ml to 5 micrograms/ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staining and of resolution versus quantification are discussed.

3T3 Cells↗
PubMed1994

Streptavidin-tricolor is a reliable marker for nonviable cells subjected to permeabilization or fixation.

Cell death is normally accompanied by loss of integrity of the cell membrane. Concomitant loss of osmotic pressure and permeability for DNA binding dyes like propidium iodide make it possible to distinguish viable from nonviable cells. However, after permeabilization for intracellular staining or after fixation of the cells, we find that propidium iodide leaks out of nonviable cells and is transferred to formerly viable cells. Cell size cannot be used for examining viability in permeabilized or heterogeneous cell populations. Here we show that streptavidin-tricolor enters specifically and irreversibly into dead cells, and is not transferred to formerly viable cells after fixation or permeabilization. Therefore, streptavidin-tricolor can be a useful dead-cell marker in experimental situations where conventional methods fail to distinguish between viable and nonviable cells.

Animals↗
PubMed2005

Quantification and functional analysis of chemotaxis by laser scanning cytometry.

BACKGROUND: Evaluation of chemotaxis assays traditionally relies on cumbersome and at times inaccurate visual counting. Moreover, many physiologic parameters that could be evaluated in conjunction with chemotactic migration, aside from morphologic changes, usually are not assessed due to the lack of a simultaneous method of analysis. We tested the suitability of laser scanning cytometry (LSC) as a convenient platform for counting migrated cells and for concurrent analysis of some features associated with their physiologic status. METHODS: We induced migration of THP-1 monocytes across Nuclepore filters with monocyte chemotactic protein-1 or vascular endothelial growth factor, alone or in combination. Filters were collected, and cells were fixed on filters and stained with the nuclear stain propidium iodide. Chemotactic indices were obtained by counting representative microscopic fields and by scanning the filters in LSC mode. RESULTS: We found an excellent correlation between direct counting and LSC. In addition, the software tools embodied in the LSC instrument allowed the observation of changes in nuclear compactness (increase in propidium iodide brightness) and morphology (increase in nuclear area and perimeter) that occurred in transmigrated cells. Monocyte chemotactic protein-1 and vascular endothelial growth factor acted as additive stimuli on these parameters. CONCLUSIONS: LSC analysis of cells undergoing chemotaxis provides a reliable and comprehensive assessment of the numbers and distribution of migrated cells and some of their nuclear parameters. The method can be easily extended to include the assessment of coincident molecular changes in cells due to chemotactic stimulation.

Cell Line↗
PubMed2005

DNA index, genome size, and electronic nuclear volume of vertebrates from the Miami Metro Zoo.

BACKGROUND: Flow cytometry is a rapid and reliable method for measuring nuclear DNA content and genome size. Fluorochrome binding characteristics, sample preparation and differences in DNA condensation, and availability of binding sites can cause variations in results obtained. METHODS: Blood samples from 82 vertebrate species were collected in 10% dimethyl sulfoxide and stained with propidium iodide/hypotonic citrate or 4,6-diamidino-2-phenylindole dihydrochloride for analysis of DNA content and electronic nuclear volume (ENV). Trout red blood cells (TRBCs), human peripheral blood lymphocytes, and human buccal cavity cells were used as internal standards. RESULTS: Mean fluorescence channel (MFC) values of TRBC and buccal cavity cells used as internal standards were stable at 15 to 120 min of propidium iodide staining. TRBCs mixed with other cells especially human peripheral blood cells showed an increase in MFC. ENV and MCF values were less variable in different species of birds than in reptiles or mammals. Genome size based on use of buccal cavity cells as the internal standard showed a high degree of correlation with previous reports. CONCLUSIONS: Proper selection and use of internal standards and sample preparation are essential for reliable determination of DNA content and genome size in vertebrates by flow cytometry.

Animals↗
PubMed2006

Whole insect and mammalian embryo imaging with confocal microscopy: morphology and apoptosis.

BACKGROUND: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays. METHODS: The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB. RESULTS: Tissues as thick as 500 microm can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide). CONCLUSIONS: The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red-emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were approximately 500-microm thick.

Amines↗