Use Of Fluorescent Protein Research Articles

Green-Emitting Rhodamine Dyes for Vital Labeling of Cell Organelles Using STED Super-Resolution Microscopy.

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining.

CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans.

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.

Self Assembled Recombinant Proteins on Metallic Nanoparticles As Bimodal Imaging Probes.

Combining multiple modalities is at the center of developing new methods for sensing and imaging that are required for comprehensive understanding of events at the molecular level. Various imaging modalities have been developed using metallic nanoparticles owning to their exceptional physical and chemical properties. Due to their localized surface plasmon resonance characteristics, gold and silver nanoparticles exhibit unique optoelectronic properties commonly used in biomedical sciences and engineering. Self assembled monolayers or physical adsorption have previously been adapted to functionalize the surfaces of nanoparticles with biomolecules for targeted imaging. However, depending on differences among the functional groups used on the nanoparticle surface, wide variation in the displayed biomolecular property to recognize its target may result. In the last decade, the properties of inorganic binding peptides have been proven advantageous to assemble selective functional nano-entities or proteins onto nanoparticles surfaces. Herein we explored formation of self-assembled hybrid metallic nano-architectures that are composed of gold and silver nanoparticles with fluorescent proteins, for use as bimodal imaging probes. We employed metal binding peptide-based assembly to self assemble green fluorescence protein onto metallic substrates of various geometries. Assembly of the green fluorescent proteins, genetically engineered to incorporate gold- or silver-binding peptides onto metallic nanoparticles, resulted in the generation of hybrid-, biomodal-imaging probes in a single step. Green fluorescent activity on gold and silver surfaces can be been monitored using both plasmonic and fluorescent signatures. Our results demonstrate a novel bimodal imaging system that can be finely tuned with respect to nanoparticle size and protein concentration. Resulting hybrid probes may mitigate the limitation of depth penetration into biological tissues as well as providing high signal-to-noise ratio and sensitivity.

Peak emission wavelength and fluorescence lifetime are coupled in far-red, GFP-like fluorescent proteins.

The discovery and use of fluorescent proteins revolutionized cell biology by allowing the visualization of proteins in living cells. Advances in fluorescent proteins, primarily through genetic engineering, have enabled more advanced analyses, including Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) and the development of genetically encoded fluorescent biosensors. These fluorescence protein-based sensors are highly effective in cells grown in monolayer cultures. However, it is often desirable to use more complex models including tissue explants, organoids, xenografts, and whole animals. These types of samples have poor light penetration owing to high scattering and absorption of light by tissue. Far-red light with a wavelength between 650-900nm is less prone to scatter, and absorption by tissues and can thus penetrate more deeply. Unfortunately, there are few fluorescent proteins in this region of the spectrum, and they have sub-optimal fluorescent properties including low brightness and short fluorescence lifetimes. Understanding the relationships between the amino-acid sequences of far-red fluorescence proteins and their photophysical properties including peak emission wavelengths and fluorescence lifetimes would be useful in the design of new fluorescence proteins for this region of the spectrum. We used both site-directed mutagenesis and gene-shuffling between mScarlet and mCardinal fluorescence proteins to create new variants and assess their properties systematically. We discovered that for far-red, GFP-like proteins the emission maxima and fluorescence lifetime have a strong inverse correlation.

Polyglutamine toxicity assays highlight the advantages of mScarlet for imaging in Saccharomyces cerevisiae.

Development of fluorescent proteins (FPs) enabled researchers to visualize protein localization and trafficking in living cells and organisms. The extended palette of available FPs allows simultaneous detection of multiple fluorescent fusion proteins. Importantly, FPs are originally derived from different organisms from jelly fish to corals and each FP displays its own biophysical properties. Among these properties, the tendency of FPs to oligomerize inherently affects the behavior of its fusion partner. Here we employed the budding yeast Saccharomyces cerevisiae to determine the impact of the latest generation of red FPs on their binding partner. We used a yeast assay based on the aggregation and toxicity of misfolded polyQ expansion proteins linked to Huntington's disease. Since polyQ aggregation and toxicity are highly dependent on the sequences flanking the polyQ region, polyQ expansions provide an ideal tool to assess the impact of FPs on their fusion partners. We found that unlike what is observed for green FP variants, yemRFP and yFusionRed-tagged polyQ expansions show reduced toxicity. However, polyQ expansions tagged with the bright synthetically engineered ymScarlet displayed severe polyQ toxicity. Our data indicate that ymScarlet might have significant advantages over the previous generation of red FPs for use in fluorescent fusions in yeast.

Polyglutamine toxicity assays highlight the advantages of mScarlet for imaging in Saccharomyces cerevisiae

Development of fluorescent proteins (FPs) enabled researchers to visualize protein localization and trafficking in living cells and organisms. The extended palette of available FPs allows simultaneous detection of multiple fluorescent fusion proteins. Importantly, FPs are originally derived from different organisms from jelly fish to corals and each FP displays its own biophysical properties. Among these properties, the tendency of FPs to oligomerize inherently affects the behavior of its fusion partner. Here we employed the budding yeast Saccharomyces cerevisiae to determine the impact of the latest generation of red FPs on their binding partner. We used a yeast assay based on the aggregation and toxicity of misfolded polyQ expansion proteins linked to Huntington’s disease. Since polyQ aggregation and toxicity are highly dependent on the sequences flanking the polyQ region, polyQ expansions provide an ideal tool to assess the impact of FPs on their fusion partners. We found that unlike what is observed for green FP variants, yemRFP and yFusionRed-tagged polyQ expansions show reduced toxicity. However, polyQ expansions tagged with the bright synthetically engineered ymScarlet displayed severe polyQ toxicity. Our data indicate that ymScarlet might have significant advantages over the previous generation of red FPs for use in fluorescent fusions in yeast.

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells.

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Polyglutamine toxicity assays highlight the advantages of mScarlet for imaging in Saccharomyces cerevisiae

Development of fluorescent proteins (FPs) enabled researchers to visualize protein localization and trafficking in living cells and organisms. The extended palette of available FPs allows simultaneous detection of multiples fluorescent fusion proteins. Importantly, FPs are originally derived from different organisms from jelly fish to corals and each FP display its own biophysical properties. Among these properties, the tendency of FPs to oligomerize inherently affects the behavior of its fusion partner. Here we employed the budding yeast Saccharomyces cerevisiae to determine the impact of the latest generation of red FPs on their binding partner. We used a yeast assay based on the aggregation and toxicity of misfolded polyQ expansion proteins linked to Huntington’s disease. Since polyQ aggregation and toxicity are highly dependent on the sequences flanking the polyQ region, polyQ expansions provide an ideal tool to assess the impact of FPs on their fusion partners. We found that unlike yemRFP and yFusionRed, the synthetically engineered ymScarlet displayed severe polyQ toxicity and aggregation similar to what is observed for green FP variants. Our data indicate that ymScarlet might have significant advantages over the previous generation of red FPs for use in fluorescent fusions in yeast.

Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling

Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio. Furthermore, imaging with longer wavelengths holds promise for reducing photostress. Here, we demonstrate that a strategy based on enzymatic self-labeling of the HaloTag fusion protein by high-performance synthetic fluorophore labels provides a robust avenue to superior in vivo analysis with STED nanoscopy in the far-red spectral range. We illustrate our approach by mapping the nanoscale distributions of the abundant scaffolding protein PSD95 at the postsynaptic membrane of excitatory synapses in living mice. With silicon-rhodamine as the reporter fluorophore, we present imaging with high contrast and low background down to ∼70-nm lateral resolution in the visual cortex at ≤25-µm depth. This approach allowed us to identify and characterize the diversity of PSD95 scaffolds in vivo. Besides small round/ovoid shapes, a substantial fraction of scaffolds exhibited a much more complex spatial organization. This highly inhomogeneous, spatially extended PSD95 distribution within the disk-like postsynaptic density, featuring intricate perforations, has not been highlighted in cell- or tissue-culture experiments. Importantly, covisualization of the corresponding spine morphologies enabled us to contextualize the diverse PSD95 patterns within synapses of different orientations and sizes.

Visualizing the Tumor Microenvironment by Color-coded Imaging in Orthotopic Mouse Models of Cancer.

The tumor microenvironment (TME) contains stromal cells in a complex interaction with cancer cells. This relationship has become better understood with the use of fluorescent proteins for in vivo imaging, originally developed by our laboratories. Spectrally-distinct fluorescent proteins can used for color-coded imaging of the complex interaction of the tumor microenvironment in the living state using cancer cells expressing a fluorescent protein of one color and host mice expressing another-color fluorescent protein. Cancer cells engineered in vitro to express a fluorescent protein were orthotopically implanted into transgenic mice expressing a fluorescent protein of a different color. Confocal microscopy was then used for color-coded imaging of the TME. Color-coded imaging of the TME has enabled us to discover that stromal cells are necessary for metastasis. Patient-derived orthotopic xenograft (PDOX) tumors were labeled by first passaging them orthotopically through transgenic nude mice expressing either green, red, or cyan fluorescent protein in order to label the stromal cells of the tumor. The colored stromal cells become stably associated with the PDOX tumors through multiple passages in transgenic colored mice or non-colored mice. The fluorescent protein-expressing stromal cells included cancer-associated fibroblasts and tumor-associated macrophages. The cancer cells in PDOX models can also be labeled with a telomerase-dependent adenovirus containing the gene for green fluorescent protein. Using this model, specific cancer-cell or stromal-cell targeting by potential therapeutics can be visualized. Color-coded imaging enabled the visualization of apparent fusion of cancer and stromal cells. Color-coded imaging is a powerful tool visualizing the interaction of cancer and stromal cells during cancer progression and treatment.

Characterization of Pax3 and Sox10 transgenic Xenopus laevis embryos as tools to study neural crest development

The neural crest is a multipotent population of cells that originates a variety of cell types. Many animal models are used to study neural crest induction, migration and differentiation, with amphibians and birds being the most widely used systems. A major technological advance to study neural crest development in mouse, chick and zebrafish has been the generation of transgenic animals in which neural crest specific enhancers/promoters drive the expression of either fluorescent proteins for use as lineage tracers, or modified genes for use in functional studies. Unfortunately, no such transgenic animals currently exist for the amphibians Xenopus laevis and tropicalis, key model systems for studying neural crest development. Here we describe the generation and characterization of two transgenic Xenopus laevis lines, Pax3-GFP and Sox10-GFP, in which GFP is expressed in the pre-migratory and migratory neural crest, respectively. We show that Pax3-GFP could be a powerful tool to study neural crest induction, whereas Sox10-GFP could be used in the study of neural crest migration in living embryos.

Fluorescent Proteins as Sensors for Cellular Behavior in Mice.

Imaging of cancer cells in mice expressing fluorescent proteins has allowed the real-time tracing of cancer growth and metastasis and determination of efficacy of candidate antitumor and antimetastatic agents, especially in mouse orthotopic models. The use of fluorescent proteins to differentially label cancer cells in the nucleus and cytoplasm can visualize the nuclear-cytoplasmic dynamics of cancer cells in vivo, including mitosis, apoptosis, cell-cycle position, and differential behavior of nucleus and cytoplasm that occurs during cancer cell deformation, migration, and extravasation. Recent applications of the technology described here include linking fluorescent proteins with cell cycle-specific proteins such that the cells change color from red to green as they transit from G1 to S phases. Any in vivo process can be imaged using fluorescent proteins, allowing molecular biology to advance from in vitro studies to studying molecular processes in the living animal.

Composite poplars: a novel tool for ectomycorrhizal research

Key MessageComposite poplars were used for ectomycorrhiza formation. Structurally normal mycorrhizas of transgenic roots revealed better fungal sugar support. Targeting fluorescent proteins to peroxisomes allowed easy in planta visualization of successful transformation.A bottle neck in ectomycorrhizal research is the time demand for generation of transgenic plants. An alternative strategy for such root-centered research might be the formation of the so-called composite plants, where transgenic roots are formed by non-transgenic shoots. We have developed an Agrobacterium rhizogenes-mediated root transformation protocol using axenic Populus tremula × tremuloides and P. tremula × alba cuttings. When comparing four different bacterial strains, A. rhizogenes K599 turned out to be the most suitable for poplar transformation. Transgenic roots revealed only minor hairy root phenotype when plants were grown on agar plates with synthetic growth medium in the absence of a sugar source. When using different ectomycorrhizal fungi, formation of ectomycorrhizas by transgenic roots of composite poplars was not affected and mycorrhizas were anatomically indistinguishable from mycorrhizas of non-transgenic roots. Elevated trehalose content and marker gene expression, however, pointed towards somewhat better fungal carbon nutrition in ectomycorrhizas of transgenic compared to non-transgenic roots. Cell wall autofluorescence of poplar fine roots is an issue that can limit the use of fluorescent proteins as visual markers for in planta analysis, especially after ectomycorrhiza formation. By targeting marker proteins to peroxisomes, sensitive fluorescence detection, easily distinguishable from cell wall autofluorescence, was obtained for both poplar fine roots and ectomycorrhizas.

Green fluorescent protein (GFP): is seeing believing and is that enough?

Intracellular compartmentalisation is a significant barrier to the successful nucleocytosolic delivery of biologics. The endocytic system has been shown to be responsible for compartmentalisation, providing an entry point, and trigger(s) for the activation of drug delivery systems. Consequently, many of the technologies used to understand endocytosis have found utility within the field of drug delivery. The use of fluorescent proteins as markers denoting compartmentalisation within the endocytic system has become commonplace. Several of the limitations associated with the use of green fluorescent protein (GFP) within the context of drug delivery have been explored here by asking a series of related questions: (1) Are molecules that regulate fusion to a specific compartment (i.e. Rab- or SNARE-GFP fusions) a good choice of marker for that compartment? (2) How reliable was GFP-marker overexpression when used to define a given endocytic compartment? (3) Can glutathione-s-transferase (GST) fused in frame with GFP (GST–GFP) act as a fluid phase endocytic probe? (4) Was GFP fluorescence a robust indicator of (GFP) protein integrity? This study concluded that there are many appropriate and useful applications for GFP; however, thought and an understanding of the biological and physicochemical character of these markers are required for the generation of meaningful data.

Fluorescent Lactic Acid Bacteria and Bifidobacteria as Vehicles of DNA Microbial Biosensors.

Control and quantification of effector molecules such as heavy metals, toxins or other target molecules is of great biotechnological, social and economic interest. Microorganisms have regulatory proteins that recognize and modify the gene expression in the presence or absence of these compounds (effector molecules) by means of binding to gene sequences. The association of these recognizing gene sequences to reporter genes will allow the detection of effector molecules of interest with high sensitivity. Once investigators have these two elements—recognizing gene sequences and reporter genes that emit signals—we need a suitable vehicle to introduce both elements. Here, we suggest lactic acid bacteria (LAB) and bifidobacteria as promising carrier microorganisms for these molecular biosensors. The use of fluorescent proteins as well as food-grade vectors and clustered regularly interspaced short palindromic repeats (CRISPR) are indispensable tools for introducing biosensors into these microorganisms. The use of these LAB and bifidobacteria would be of special interest for studying the intestinal environment or other complex ecosystems. The great variety of species adapted to many environments, as well as the possibility of applying several protocols for their transformation with recognizing gene sequences and reporter genes are considerable advantages. Finally, an effort must be made to find recognizable gene sequences.

Strategies for In Vivo Imaging Using Fluorescent Proteins.

Fluorescent proteins have enabled the color-coding of cells growing in vivo. Noninvasive imaging of cells expressing fluorescent proteins has allowed the real-time determination of the behavior of cancer cells, the progression of infection, the differentiation of stem cells, and interaction of stromal and cancer cells. Cancer cells labeled in the nucleus and cytoplasm with spectrally-distinct proteins can visualize in vivo nuclear-cytoplasmic dynamics in vivo including: mitosis, apoptosis, cell-cycle phase, and differential behavior of nucleus and cytoplasm that occurs during cancer-cell deformation. Linking spectrally-distinct fluorescent proteins with cell-cycle-specific proteins results in color-coding the phases of the cell cycle. With the use of fluorescent proteins, literally any cellular or molecular function can be imaged in vivo. J. Cell. Biochem. 118: 2571-2580, 2017. © 2017 Wiley Periodicals, Inc.

Purification and Characterization of Clover‐Ruby2 as a FRET Substrate for Protease Assays

The use of fluorescent proteins (FPs) in biomedical research has significantly expanded our ability to monitor biological processes in real‐time. These processes include protein expression, localization, cellular trafficking, ligand binding, and other biological events where development of imaging methodology is still underway. While the green fluorescent protein (GFP) was the first molecule in this class to be discovered, there is currently a wide range of FPs available that vary in their spectroscopic, physical, and chemical properties. Förster (fluorescence) resonance energy transfer (FRET) is one area of biological imaging that relies on a donor FP and acceptor FP to facilitate the transfer of energy. While the cyan and yellow FPs have been more commonly used as a FRET pair, green and red FPs offer some advantages for live cell FRET imaging, such as lower phototoxicity, lower autofluorescence, and greater spectral separation. We have investigated the Clover‐Ruby2 protein as a substrate FRET pair for monitoring protease activity in both in vitro and in vivo applications. This is aligned with the goal of developing an in vitro model system to mimic protease events occurring in live cells. The Clover (C) and Ruby2 (R2) proteins have been expressed and purified from E. coli, both as individual proteins (C and R2), and as a tandem FRET pair (CR2) linked through a protease recognition region. To obtain optimal samples for these studies, purification strategies have been optimized using immobilized‐metal‐affinity‐, ion‐exchange‐, hydrophobic‐, and gel filtration‐chromatography. Characterization of the CR2 fusion protein using UV‐Vis absorbance and fluorescence spectroscopy at various steps during the purification procedure have provided results demonstrating the quality of FRET signal being dependent upon sample purity, salt concentration, and other factors that are still being investigated. Maturation of R2 was found to be most critical for obtaining quality dynamic FRET measurements, and it appears the level of R2 maturation achieved is also influenced by its chemical environment. Additionally, R2 was found to be become much more hydrophobic following the maturation process, as observed during hydrophobic chromatography (HC) separation where the non‐mature R2 showed very low affinity for HC media, and matured R2 had much greater affinity. Finally, gel filtration experiments demonstrated a significant degree of interaction of CR2 with free‐C and/or free–R2. This raised some concerns about using these proteins for FRET assays where the dynamic properties of the FRET signal is used to determine kinetic rates. If C and R2 interactions show an increase in FRET simply due to hydrophobicity, and non specific interactions, the FRET signal will be artificially increased, and the C and R2 pair may not dissociate in real‐time following the protease reaction. To overcome these limitations, we have developed a number of calibration procedures, obtained under different chemical conditions, to monitor the assays. This allowed normalization of the assays in different reaction environments, which is critical when working with proteases that have optimal activity under varied chemical reaction conditions.Support or Funding InformationNIH R15 GM080691 02

Intracellular Delivery of Membrane Impermeable Photostable Fluorescent Probes into Living Cells for Super-Resolution Microscopy

Specific labeling of the cellular target with fluorophore is one of the fundamental requirement in all fluorescence imaging techniques including single molecule and super-resolution microscopy techniques. While extracellular targets may be tagged with virtually any kind of probes, intracellular labeling of living cells is limited to the use of fluorescent proteins and limited selection of membrane permeable dyes. Here we show that pore forming proteins can be used to temporarily permeabilize the cells and allow delivery of various fluorescent probes, ranging from organic dyes (<1 kDa) to fluorescent immunoglobulin antibody (∼150 kDa), for specific labeling of intracellular targets for live fluorescence cell imaging. We demonstrate that permeabilized cells can efficiently be recovered to carry on normal cellular processes shown by nuclear translocation of nanobody-labeled p65 proteins in response to chemical stimulation. One of the most photostable but membrane impermeable organic fluorophore, Atto 647N, has been delivered into cells to label actin fibers and kinesin dimers and to perform super-resolution fluorescence microscopy imaging dSTORM and single molecule tracking, respectively. This technique opens up a large number of stable, but otherwise membrane impermeable fluorescence labeling probes that are available for investigating transfected and endogenous intracellular targets.

Reflection imaging of China ink-perfused brain vasculature using confocal laser-scanning microscopy after clarification of brain tissue by the Spalteholz method.

Confocal laser-scanning microscopy is a useful tool for visualizing neurons and glia in transparent preparations of brain tissue from laboratory animals. Currently, imaging capillaries and venules in transparent brain tissues requires the use of fluorescent proteins. Here, we show that vessels can be imaged by confocal laser-scanning microscopy in transparent cortical, hippocampal and cerebellar preparations after clarification of China ink-injected specimens by the Spalteholz method. This method may be suitable for global, three-dimensional, quantitative analyses of vessels, including stereological estimations of total volume and length and of surface area of vessels, which constitute indirect approaches to investigate angiogenesis.