Optimal processing method to obtain four-color confocal fluorescent images of the cytoskeleton and nucleus in three-dimensional chondrocyte cultures.

Tissue engineering of articular cartilage requires accurate imaging of the chondrocyte cytoskeleton. Past studies have applied various fixation and permeabilization protocols without optimization of parameters. In this study, we have examined procedures using glutaraldehyde and paraformaldehyde as fixatives and Triton X-100 and Octyl-POE as permeabilizing detergents. A four-color fluorescence confocal method was developed to simultaneously image actin, tubulin, vimentin, and the nucleus. We found optimal preservation and morphology of the chondrocyte cytoskeleton after simultaneous fixation and permeabilization with glutaraldehyde and Triton X-100. These images displayed less cellular shrinkage and higher-resolution filamentous structures than with paraformaldehyde or when permeabilization followed fixation.     

Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins

The number of proteins known to be associated with lipid droplets (LDs) is increasing. However, the reported distribution of a given protein in the LDs was, in some cases, found not reproduced by other groups. We report here that the choice of the fixation and permeabilization method is important in order to observe LD proteins using immunofluorescence microscopy. Formaldehyde fixation followed by treatment with Triton X-100, one of the most frequently used protocols for the immunolabeling of cultured cells, was not appropriate to label adipocyte differentiation-related protein (ADRP), TIP47, or Rab18 in LDs. Formaldehyde fixation followed by treatment with digitonin or saponin, allowed the visualization of all these proteins in LDs. When cells were fixed with glutaraldehyde, permeabilization by Triton X-100 could also be used for ADRP. These observations suggest that LD proteins are likely to be solubilized by some detergents, and strong cross-linkage to the surrounding protein matrix or mild permeabilization is necessary for their retention on the LD surface. The authors Yuki Ohsaki and Takashi Maeda have contributed equally to this work.     

Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.     

Three new Drosophila markers of intracellular membranes

The need for cellular markers that permit a quick and accurate evaluation of a protein's subcellular localization has increased with the surge of new data generated by the Drosophila genome project. In this report, we present three ubiquitously expressed Drosophila transgenes that expressed a green fluorescent protein variant (enhanced yellow fluorescent protein) that has been targeted to different intracellular membrane targets: the Golgi apparatus, mitochondria, and endoplasmic reticulum. These markers serve as an internal standard for characterizing a protein's subcellular localization or as a means of tracking the dynamics of intracellular organelles during normal or abnormal cellular or developmental processes. We have also examined fixation artifacts using these constructs to illustrate the effects that fixation and permeabilization have on intracellular membrane organization.     

Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells

Permeabilization was evaluated as a rapid method to prepare mammalian cells for subcellular enzyme activity measurement. It was observed that enzymes can be measured directly in cell suspensions permeabilized by Triton X-100 and digitonin with various concentrations. Total enzyme activities measured in permeabilized cells were identical to those measured in sonicated cells showing that permeabilization can replace the more complicated sonication method. Tuning of digitonin concentration allowed selective permeabilization of plasma and mitochondrial membranes. This was studied by analyzing the release of extramitochondrial and mitochondrial marker enzymes on treatment with different concentrations of the agent. Solely the plasma membrane was permeabilized by using 0.01–0.02% (w/v) digitonin. Access to all cellular enzymes was achieved by using 0.05% (v/v) Triton X-100. This selective permeabilization was further evaluated in a 96-well plate format by testing additional marker enzymes and additional cell lines, Hep G2 and CHO-K1, applying the developed protocol. The presented method is well suited for the high-throughput analysis of subcellular localization and activity of enzymes. The method is simple and enables one to distinguish between mitochondrial and extramitochondrial activities, which is usually achieved only by much more complicated and time-consuming cell preparation.     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

Fluorescence Tracing of Intracellular Proteins

Developments in fluorescence microscopy and the availability of fluorescently labeled antibodies and probes for localization of molecules and organelles have made the microscope an indispensable tool with which one can map specific molecules to subcellular loci allowing deep insight into cell and organelle biology. Furthermore, confocal microscopy permits analysis of the three dimensional architecture of cells that could not be accomplished by conventional light microscopy. The goal of fluorescence protein tracing by microscopy is to visualize cellular constituents and general cytoarchitecture as close to native organization as possible. To achieve this, and to preserve cellular structure in the best possible manner, the specimen is usually fixed chemically. Here I review several standard fixation, permeabilization and labeling schemes followed by examples of several standard imaging techniques.     

Expression of a novel sodium-hydrogen exchanger in the gastrointestinal tract and kidney

Aquaporin CHIP, a 28 kDa channel forming protein, has been proposed to function as water channel in both erythrocyte and kidney proximal tubule. Recently, we have reported that in frog urinary bladder, a model of the kidney collecting tubule, polyclonal antibodies against human erythrocyte CHIP recognize and immunoprecipitate a 30 kDa protein from the epithelial cell homogenate. In the present work confocal fluorescence microscopy was used to determine the cellular and subcellular localization of CHIP28-like proteins in the urinary epithelium. A clear labeling of the apical border was found after Triton X-100 permeabilization. The labeling was distributed throughout the apical domain and not restricted to specific domains of the membrane. The staining was also present in the deeper confocal sections where the fluorescence seems to be localized at the cellular contour. No difference in the labeling patterns was observed between resting and ADH-treated bladder. Specificity of the staining was confirmed by the absence of the labeling pattern when antiserum was preadsorbed on CHIP28 protein immobilized on Immobilon P stripes. Our results suggest that CHIP-like proteins are not proteins inserted in the apical membrane during the antidiuretic response. Moreover, we do not know whether the labeling was due to the presence of CHIP28 itself or an as-yet-unidentified protein sharing immunological analogies with aquaporin CHIP.     

Detection of S-100b protein in Triton cytoskeletons: an immunocytochemical study on cultured Schwann cells

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.     

Microfilament distribution and adhesion patterns in cultured cells after glutaraldehyde-formaldehyde fixation

To study the relationship between microfilament distribution and adhesion patterns in the same cultured cell, we have employed a simple glutaraldehyde-formaldehyde fixation technique followed by permeabilization of the cells in buffered Triton X-100. This method gives an excellent preservation of cellular morphology in general and of adhesion patterns in particular for examination with surface reflection interference microscopy. It also permits the concomitant use of the actin-specific fluorescent probe NBD-phallacidin to visualize the distribution of microfilaments.     

Combining patch-clamping of cells in brain slices with immunocytochemical labeling to define cell type and developmental stage

In neuroscience, combining patch-clamping with protein identification in the same cell is becoming increasingly important to define which subtype or developmental stage of a neuron or glial cell is being recorded from, and to attribute measured membrane currents to expressed ion channels or receptors. Here, we describe a protocol to achieve this when studying cells in acute brain slices, which antibodies penetrate poorly into and for which detergent permeabilization cannot be used when using antibodies that recognize lipid components such as O4 sulfatide. The method avoids the need for resectioning of the electrophysiologically recorded slices. It employs filling of the cell with a fluorescent dye during whole-cell recording, to allow subsequent localization of the cell, followed by fixation and free-floating section labeling with up to three antibodies, which may recognize membrane, nuclear or cytosolic proteins. With practice, approximately 80% of patch-clamped cells can be retrieved and have their proteins identified in this way. The entire protocol can be completed in 3-4 d.     

Differential localization of von Willebrand factor, fibronectin and 13-HODE in human endothelial cell cultures

Von Willebrand factor (vWF), fibronectin (FN) and 13-hydroxy-octadecadienoic acid (13-HODE) are known to influence the regulation of the adhesive properties of vascular surfaces. In the present study vWF, FN and 13-HODE were comparatively localized in endothelial cells (EC) and in the extracellular matrix (ECM) produced by EC. An indirect immunofluorescent technique was applied to coverslips containing human EC cultures previously fixed and permeabilized following different procedures: A. Alcohol/acetone; B. Paraformaldehyde alone and C. Paraformaldehyde followed by Triton X-100. vWF was observed inside EC (A), on the ECM produced by EC (B) or in EC and ECM (C) depending on the fixation procedures used. FN was mainly localized in the ECM despite the fixation procedures employed. FN was only seen in relation to cell bodies after strong permeabilization (A). Under our experimental conditions 13-HODE was never found in ECM. This latter antigen was observed randomly dispersed in those preparations fixed with alcohol/acetone, indicating that it is probably extracted by this fixative. 13-HODE was detected in granular shaped structures in EC after permeabilization with detergent (C). These results suggest that the cellular localization of vWF and FN is compatible with an adhesive role related to the abluminal side of ECs. 13-HODE was readily observed after mild permeabilization. This finding would be morphologically consistent with its contribution to the regulation of the vessel wall thromboresistance.     

Evaluation of VP22 spread in tissue culture

We compare methods of detection of intercellular transport of the herpes simplex virus protein VP22 and of a green fluorescent protein (GFP)-VP22 fusion protein. Spread of both proteins was observed by immunofluorescence (IF) using organic fixatives. Spread of both proteins was also detected by IF after paraformaldehyde (PFA) fixation and detergent permeabilization, albeit at reduced levels. However, while spread of GFP-VP22 was observed by examining intrinsic GFP fluorescence after methanol fixation, little spread was observed after PFA fixation, suggesting that the levels of the fusion protein in recipient cells were below the detection limits of intrinsic-fluorescence or that PFA fixation quenches the fluorescence of GFP-VP22. We further considered whether elution of VP22 from methanol-fixed cells and postfixation binding to surrounding cells contributed to the increased detection of spread observed after methanol fixation. The results show that while this could occur, it appeared to be a minor effect not accounting for the observed VP22 cell-to-cell spread in culture.     

Paraformaldehyde Fixation May Lead to Misinterpretation of the Subcellular Localization of Plant High Mobility Group Box Proteins

Arabidopsis High Mobility Group Box (HMBG) proteins were previously found associated with the interphase chromatin but not the metaphase chromosome. However, these studies are usually based on immunolocalization analysis involving paraformaldehyde fixation. Paraformaldehyde fixation has been widely adapted to preserved cell morphology before immunofluorescence staining. On one hand, the processed cells are no longer living. On the other hand, the processing may lead to misinterpretation of localization. HMGBs from Arabidopsis were fused with enhanced green fluorescence protein (EGFP) and transformed into tobacco BY-2 cells. Basically, the localization of these HMGB proteins detected with EGFP fluorescence in interphase agreed with previous publications. Upon 4% paraformaldehyde fixation, AtHMGB1 was found associated with interphase but not the metaphase chromosomes as previously reported. However, when EGFP fluorescence signal was directly observed under confocal microscope without fixation, association of AtHMGB1 with metaphase chromosomes can be detected. Paraformaldehyde fixation led to dissociation of EGFP tagged AtHMBG1 protein from metaphase chromosomes. This kind of pre-processing of live specimen may lead to dissociation of protein-protein or protein-nucleic acid interaction. Therefore, using of EGFP fusion proteins in live specimen is a better way to determine the correct localization and interaction of proteins.     

蛋白质通量化定位体系的建立及初步应用

目的:对于蛋白质功能而言,蛋白质定位与蛋白质的表达和修饰等同等重要。传统的蛋白质定位一直沿用单个基因、逐个的研究方法,本实验拟建立一种通量蛋白质定位研究体系。方法:采用并优化了细胞微阵列技术,结合绿色荧光蛋白(GFP)标签、激光扫描共聚焦显微镜及反转染技术,用于大规模蛋白质定位研究。结果:初步建立的蛋白质定位微阵列包含107个GFP标记的cDNA表达载体,分别编码107个重要细胞信号传导通路的蛋白质,并与定位数据库中的已知结果进行了比对;对该系统的有效性进行了验证评价。结论:本定位系统可有效地用于通量化蛋白质定位研究,并可以发展用于蛋白质相互作用、泛素-蛋白酶体通路底物筛选等进一步的功能研究。     

Migration of bone marrow stromal cells in 3D: 4 color methodology reveals spatially and temporally coordinated events

The cytoskeleton plays a central role in many cell processes including directed cell migration. Since most previous work has investigated cell migration in two dimensions (2D), new methods are required to study movement in three dimensions (3D) while preserving 3D structure of the cytoskeleton. Most previous studies have labeled two cytoskeletal networks simultaneously, impeding an appreciation of their complex and dynamic interconnections. Here we report the development of a 4 color method to simultaneously image vimentin, actin, tubulin and the nucleus for high-resolution confocal microscopy of bone-marrow stromal cells (BMSCs) migrating through a porous membrane. Several methods were tested for structural preservation and labeling intensity resulting in identification of an optimized simultaneous fixation and permeabilization method using glutaraldehyde, paraformaldehyde and Triton X-100 followed by a quadruple fluorescent labeling method. This procedure was then applied at a sequence of time points to migrating cells, allowing temporal progression of migration to be assessed by visualizing all three networks plus the nucleus, providing new insights into 3D directed cell migration including processes such as leading edge structure, cytoskeletal distribution and nucleokinesis. Colocalization of actin and microtubules with distinct spatial arrangements at the cellular leading edge during migration, together with microtubule axial polarization supports recent reports indicating the pivotal role of microtubules in directed cell migration. This study also provides a foundation for 3D migration studies versus 2D studies, providing precise and robust methods to attain new insights into the cellular mechanisms of motility.