Vibratome Sections Research Articles

Morphologic Characteristics of the Submandibular Salivary Gland of the Collared Peccary (Tayassu Tajacu)

Introduction Most salivary glands is located on the inside and around the oral cavity, and are divided into major and minor salivary glands. The aim of the present study was to describe the structural and ultrastructural morphological characteristics of the lingual tissue of the submandibular glands of the collared peccary (Tayassu tajacu). Materials and Methods The submandibular glands (n = 10) of adult male collared peccaries ( T. tajacu) were used for histological and ultrastructural analysis. The techniques used were light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results The submandibular salivary glands of the collared peccary (T. tajacu) showed a capsule formed by a connective tissue containing the acinus and duct cells. Histologically, the nuclei located at the basal region of the cells was observed. The light polarized microscopy clearly showed the presence of type I and type III collagen. In the SEM image, the submandibular salivary gland revealed a round aspect separated in several lobules with bundles of collagen fibers. The vibratome sections showed the groupings of acinar cells, with intermingled secretory ducts containing vessels of different diameters. The secretory granules were noted in the apical portion of the acinar and ductal cells. The thick bundles of collagen fibers formed a glandular capsule and were identified around of the acinar and ductal cells in three-dimensional SEM images. The TEM images showed a number of secretory granules, especially in the apical region of the cytoplasm of the acinar cells and in the basal portion of the nuclei. The granular endoplasmic reticulum area, the euchromatic nuclei and the cytoplasmic projections may be seen. Mucous acinar cells separated by fine collagen fibers were also observed. Conclusion The morphological characteristics of the submandibular gland of the collared peccary is similar to that of other mammals with the same eating habits and habitat.

CD99L2 deficiency inhibits leukocyte entry into the central nervous system and ameliorates neuroinflammation.

Leukocyte entry into the CNS is a crucial step in the development of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Adhesion molecules mediating the docking of leukocytes to the endothelium of the blood-brain barrier (BBB) represent valuable targets for interference with the disease. However, little is known about the adhesion and signaling mechanisms in endothelial cells that mediate the diapedesis through the BBB. Here, we show that conditional Tie-2-Cre driven gene inactivation of CD99L2 inhibits leukocyte entry into the CNS during active MOG35-55 -induced EAE and alleviates severity of the disease. No detrimental effect on the immune response was observed. The number of perivascular cuffs around vessels of the CNS was reduced, as was the number of inflammatory foci, sites of demyelination and expression levels of pro-inflammatory cytokines. Three-dimensional analysis of vibratome sections of the CNS revealed an accumulation of leukocytes between endothelial cells and the underlying basement membrane, whereas leukocyte docking to the luminal surface of the endothelium of the BBB was unaffected. Collectively, these results suggest that CD99L2 participates in the development of EAE by supporting diapedesis of leukocytes through the endothelial basement membrane of blood vessels of the BBB in the CNS.

Characterization of Patient‐Derived Glioblastoma Stem Cells for Marker Expression, Motility, and Invasiveness

No effective treatment exists for Glioblastoma Multiforme (GBM), the most common and deadly form of brain cancer. Many studies have increasingly pointed towards Glioblastoma Stem Cells (GSCs) as the main cause of GBM malignancy, but mechanisms controlling their invasiveness are not fully understood. Our lab has shown that the L1 Cell Adhesion Molecule (L1CAM) increases GBM cell motility and proliferation through autocrine/paracrine stimulation in vitro. Our objective was to determine whether or not L1CAM plays a similar role in GSCs derived from patient surgical specimens. Our recent focus has been on characterizing our GSC lines for L1CAM expression and on developing bright fluorescently labeled GSCs for in vivo invasion experiments. Characterization of GSC lines for two accepted GSC “markers” has revealed that GSC lines isolated by our adherent culture method resulted in cells almost exclusively positive for Sox‐2 and nestin by flow cytometry. When placed in “differentiation media” containing 10% FBS, one GSC line retained stable expression of GSC markers, while another showed total loss of Sox‐2, but kept nestin. Analysis via western blot showed several GSC cell lines expressed L1CAM as well as the ADAM10 protease that cleaves L1 from the cell surface to stimulate nearby cells. GSCs infected with the K72 lentiviral vector encoding Green Fluorescent Protein (GFP) resulted in bright green fluorescent labeling. Injection of labeled GSCs was done into chick embryo brains at E5 either by themselves or mixed with other cell lines labeled with mCherry. Tumors were allowed to form for 10 days, brains were dissected and fixed at E15, and 350 um vibratome sections were mounted on slides for analysis by confocal microscopy. Analyzed were tumor formation, extent of cell invasion, and deposition of “trails” of L1CAM that might guide GSCs to be more invasive. The GSC line that exhibited stable Sox‐2 expression was highly invasive into brain, kept its stem cell morphology, and tended to form a medulla in mixed tumors with other GBM cell lines. The GSC line that lost Sox‐2 expression was also highly invasive, but many cells exhibited long processes like axons, and cells tended to form the cortex of mixed tumors with other GBM cell lines. We conclude that our GSC lines show fundamentally different invasive behavior and propensities to remain as GSCs. Furthermore, the chick embryo brain tumor model appears to allow a range of individual cell phenotypes to develop from GSC and GBM cell lines that presumably reflect their origins and, thus, it is a good model system to characterize phenotypes of patient‐derived GSC lines.Support or Funding InformationWe thank the UD Summer Scholars Program, the UD Undergraduate Research Program, Dept. of Biological Sciences Undergraduate Research Fund, and graduate student Reetika Dutt.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elucidating the physiological role of platelet‐derived growth factor receptor‐alpha+ cells and characterization of ANO1 in the murine upper urinary tract.

Spontaneous electrical activity is necessary for driving peristalsis in the upper urinary tract (UUT) and is hypothesized to originate from the proximal renal pelvis. However, the mechanisms by which auto‐rhythmicity is generated remain undetermined. Specialized “atypical” smooth muscle cells have been implicated in pacing the pelvis due to the higher abundance of these cells in the area in which spontaneous contractions are generated. The calcium‐activated chloride channel (CaCC), anoctamin‐1 (ANO1) is essential for slow wave generation and normal gastrointestinal motility. In the pelvis, NFA‐sensitive Cl− currents have been observed in interstitial cells. To investigate the role of ANO1 in the renal pelvis, quantitative RT‐PCR analysis of Ano1 expression in the renal pelvis was assessed in mRNA extracted from single cell populations. Ano1 mRNA was detected in smooth muscle myosin heavy chain‐positive (smMHC+) cells, but was present more abundantly in platelet‐derived growth factor receptor alpha‐positive (PDGFRα+) cells. Furthermore, isolated Ano1eGFP+ cells confirmed the presence of cells co‐expressing both ANO1 and PDGFRα (~95%). Immunohistochemistry further validated this as coronal sections derived from the proximal renal pelvis exhibited dense ANO1 labeling on PDGFRα+ cells. An extensive network of PDGFRα+ cells populated the width of the proximal pelvic wall except the urothelium. Interestingly, smMHC+ “atypical” cells within this proximal region also co‐expressed PDGFRα+, identifying a novel marker of these specialized smooth muscle cells. Next, to determine if Ano1 possessed a physiological role in the pelvis, edge‐tracking and Ca2+ imaging experiments were performed on intact, renal pelvic tissue. Pharmacological inhibition of ANO1 with the CaCC inhibitor, CaCCinh‐A01 (5–10 μM) reduced the frequency of contractions and propagating Ca2+ waves in typical smooth muscle cells, indicating that Ano1 may be responsible for the generation of contractions. To probe underlying intracellular Ca2+ signals in PDGFRα+ and smMHC+ cells, confocal imaging experiments were conducted on the proximal extensions of the renal pelvis from PDGFRα+‐GCaMP6f mice on coronal vibratome sections (~150 μm thick) and flat‐sheet tissue. High frequency, spontaneous intracellular Ca2+ transients were visualized within PDGFRα+/smMHC+ specialized cells, that were abolished upon removal of extracellular Ca2+ but insensitive to L‐Type Ca2+ channel blockade by nicardipine (1 μM). PDGFRα+ cells presented irregular, often long duration Ca2+ transients that occasionally propagated into neighboring cells. Taken together, these data suggest a physiological role for PDGFRα+ cells, whereby activation of ANO1 channels, expressed on their plasma membrane, could contribute to depolarizing neighboring smMHC+ cells and ultimately regulate the propagating contractions in the renal pelvis.Support or Funding InformationR01 DK‐091336 (KMS)P01‐DK041315 (KMS)P30‐GM110767This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Strong carcinogenic stress response induction of preneoplastic cells positive for GST-P in the rat liver: Physiological mechanism for initiation

AimsTo identify experimental conditions that induce preneoplastic cells positive for glutathione S-transferase P-form (GST-P) in the rat liver by new approaches, and analysis of the mechanism of cancer initiation based on the findings. Main methodsThe experimental protocols employed to induce GST-P+ preneoplastic cells in rat liver were as follows. Protocol 1: adult rats were fed basal diet containing 2-acetylaminofluorene (AAF, 0.02% by wt) and high concentrations of N-acetyl-l-cysteine (0.5%) over 10 weeks. Protocol 2: rats were subjected to partial hepatectomy (2/3PH), followed by an AAF (0.04%) diet for two more weeks. Vibratome-prepared liver sections were then immunostained for GST-P. Key findingsGST-P was inducible in the rat liver in response to the strong carcinogenic stress by AAF in the two experimental protocols. When examined immunocytochemically with vibratome sections, the biliary tracts of hepatocytes, GST-P+ single hepatocytes and foci were heavily positive for the marker enzyme in addition to ordinary cytosolic staining of preneoplastic cell populations. The biliary tracts of hepatocytes were severely injured, and the excretory portions of GST-P+ single hepatocytes were significantly injured. SignificanceThe cytotoxic action of AAF that give rise to the GST-P+ single hepatocytes was suggested to be an injury to the excretory pump(s) and the duct of hepatocytes. A new physiological mechanism was hypothesized for the induction of preneoplastic cell populations in the rat liver instead of a genetic mechanism.

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy.

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of these three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy.

Las colecciones biológicas: Los tesoros escondidos de un país mega-diverso

The gastrula morphology of Ceratophrys stolzmanni (the Pac-man frog) was studied. Its eggs, whose diameter at the beginning of the gastrulation process is 2.2 mm in diameter, develop rapidly. The gastrulation process requires five hours approximately. The embryos were fixed and analyzed in whole-mount, in bisections, and vibratome sections. The eggs of C. stolzmanni were pigmented, as in Xenopus laevis, but their size was bigger. The gastrula morphology of C. stolzmanni is similar to the one described for Ceratophrys ornata (the argentine horned-frog) by other authors. The late gastrula and early neurula stages, studied with vibratome sections stained for cell nuclei, revealed lack of endoderm covering both the posterior notochord and the somites found on this region. This region is called the GRP (Gastrocoel roof plate) and it plays a major role in the development of the left-right axis in amphibians. In this work the GRP of C. stolzmanni was characterized at the morphological level.

Cutting Thick Sections Using a Vibratome.

Sectioning with a vibrating microtome (vibratome) is a valuable procedure for generating thick sections that can be used for immunohistochemistry and in situ hybridization. It is particularly useful for revealing histological and 3D detail in tissues and embryos that have been subjected to various whole-mount histological procedures such as β-galactosidase and alkaline phosphatase staining, and fluorescent and DAB (diaminobenzidine) immunostaining. Vibratome sectioning does not involve any harsh organic solvents and is therefore suited for processing specimens stained with fluorescent antibodies or dyes. In addition, live tissue can be sectioned for subsequent culture, manipulation, and live imaging. Very few materials are required for vibratome sectioning, and it is a relatively straightforward procedure that is quickly mastered. This protocol describes basic vibratome sectioning that can be used for any staining procedure typically used in histology.

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish.

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-β-Hydroxysteroid dehydrogenase /Δ5-4 isomerase (3β-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3β-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3β-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

Isolation, culture and characterization of primary mouse RPE cells.

Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1-2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD).

A FRAP-Based Method for Monitoring Molecular Transport in Ciliary Photoreceptor Cells In Vivo.

The outer segment of rod and cone photoreceptor cells represents a highly modified primary sensory cilium. It renews on a daily basis throughout lifetime and effective vectorial transport to the cilium is essential for the maintenance of the photoreceptor cell function. Defects in molecules of transport modules lead to severe retinal ciliopathies. We have recently established a fluorescence recovery after photobleaching (FRAP)-based method to monitor molecular trafficking in living rodent photoreceptor cells. We irreversibly bleach the fluorescence of tagged molecules (e.g. eGFP-Rhodopsin) in photoreceptor cells of native vibratome sections through the retina by high laser intensity. In the laser scanning microscope, the recovery of the fluorescent signal is monitored over time and the kinetics of movements of molecules can be quantitatively ascertained.

한국관박쥐 망막의 신경전달물질 및 수용체, 뮬러세포 동정

목적: 본 연구에서는 한국관박쥐 망막에서의 시각계를 이해하기 위하여 한국관박쥐의 망막 내 글루타메이트 및 <TEX>${\gamma}$</TEX>-aminobutyric acid (GABA), 아세틸콜린과 같은 중추신경계의 주요 신경전달물질과 수용체, 신경교세포인 뮬러세포의 분포를 분석하였다. 방법: 성체 한국관박쥐의 망막을 대상으로 하였다. 망막을 수직 절편한 다음, 표준면역세포화학법을 적용하였다. 공초점 현미경을 사용하여 면역형광이미지 내 면역반응성을 확인하였다. 결과: 한국관박쥐의 망막에서 글루타메이트에 대한 면역반응성을 나타내는 신경세포들은 주로 신경절세포층에 존재하였다. GABA에 대한 면역반응성을 가지는 신경세포들은 내핵층에 주요하게 분포했으며, GABA의 수용체들은 내망상층에 존재하였다. 아세틸콜린의 면역반응성 신경세포들은 주로 내핵층에 위치하고 있었으며, 니코틴성 아세틸콜린 수용체 각각의 면역반응성들은 대부분 내망상층에 밀집해 있었다. 한국관박쥐의 망막에서 신경교세포 중 하나인 뮬러세포는 신경절 세포층에서 외핵층까지 길게 뻗어 있었다. 결론: 본 연구를 통하여 한국관박쥐의 망막에도 다른 포유동물의 망막에 있는 주요 신경전달물질 및 수용체, 뮬러세포가 존재한다는 것이 밝혀졌다. 이와 같은 연구결과는 한국관박쥐는 조직화된 망막 신경회로를 가지는 기능적 망막을 가지고 있음을 의미한다. Purpose: The objective of this study was to investigate the visual system of the greater horseshoe bat (Rhinolophus ferrumequinum) by location analysis of some major neurotransmitters glutamate, <TEX>${\gamma}$</TEX>-aminobutyric acid (GABA), acetylcholine, and their receptors, and <TEX>$m{\ddot{u}}ller$</TEX> glial cells in retina. Methods: Standard immunocytochemical techniques were used after vibratome section of retinal tissues of adult greater horseshoe bat for this study. Immnoreactions in immunofluorescence images were analyzed using confocal microscope. Results: Anti-glutamate-immunoreactive neurons were mainly localized in the ganglion cell layer (GCL). The majority of anti-GABA-immunoreactive cells distributed in the inner nuclear layer (INL), and GABAA receptors were localized in the inner plexiform layer (IPL). Anti-choline acetyltransferase-immuoreactive cholinergic neurons were mainly located in the INL and GCL, and most of nicotinic acetylcholine receptors were localized in the IPL. The <TEX>$m{\ddot{u}}ller$</TEX> cells in the retina of the greater horseshoe bat stretched theirs range from the GCL to outer nuclear layer (ONL). Conclusions: This study revealed that the retinas of the greater horseshoe bats contain the same major neurotransmitters and receptors, and glial cell in visually functional mammalian retinas. The present results may suggest that the greater horseshoe bats have the functional retinas for visual analysis through the organized retinal neural circuits.

Automated in situ brain imaging for mapping the Drosophila connectome

Mapping the connectome, a wiring diagram of the entire brain, requires large-scale imaging of numerous single neurons with diverse morphology. It is a formidable challenge to reassemble these neurons into a virtual brain and correlate their structural networks with neuronal activities, which are measured in different experiments to analyze the informational flow in the brain. Here, we report an in situ brain imaging technique called Fly Head Array Slice Tomography (FHAST), which permits the reconstruction of structural and functional data to generate an integrative connectome in Drosophila. Using FHAST, the head capsules of an array of flies can be opened with a single vibratome sectioning to expose the brains, replacing the painstaking and inconsistent brain dissection process. FHAST can reveal in situ brain neuroanatomy with minimal distortion to neuronal morphology and maintain intact neuronal connections to peripheral sensory organs. Most importantly, it enables the automated 3D imaging of 100 intact fly brains in each experiment. The established head model with in situ brain neuroanatomy allows functional data to be accurately registered and associated with 3D images of single neurons. These integrative data can then be shared, searched, visualized, and analyzed for understanding how brain-wide activities in different neurons within the same circuit function together to control complex behaviors.

A Method for Preparing Difficult Plant Tissues for Light and Electron Microscopy.

Although the advent of microwave technologies has both improved and accelerated tissue processing for microscopy, there still remain many limitations in conventional chemical fixation, dehydration, embedding, and sectioning, particularly with regard to plant materials. The Proteaceae, a family of plants widely distributed in the Southern Hemisphere and well adapted to harsh climates and nutrient-poor soils, is a perfect example; the complexity of Proteaceae leaves means that almost no ultrastructural data are available as these are notoriously difficult to both infiltrate and section. Here, a step-by-step protocol is described that allows for the successful preparation of Banksia prionotes (Australian Proteaceae) leaves for both light and transmission electron microscopy. The method, which applies a novel combination of vibratome sectioning, microwave processing and vacuum steps, and the utilization of an ultra low viscosity resin, results in highly reproducible, well-preserved, sectionable material from which very high-quality light and electron micrographs can be obtained. With this, cellular ultrastructure from the level of a leaf through to organelle substructure can be studied. This approach will be widely applicable, both within and outside of the plant sciences, and can be readily adapted to meet specific sample requirements and imaging needs.

Increased apoptosis and hypomyelination in cerebral white matter of macular mutant mouse brain

Hypomyelination in developing brain is often accompanied by congenital metabolic disorders. Menkes kinky hair disease is an X-linked neurodegenerative disease of impaired copper transport, resulting from a mutation of the Menkes disease gene, a transmembrane copper-transporting p-type ATPase gene (ATP7A). In a macular mutant mouse model, the murine ortholog of Menkes gene (mottled gene) is mutated, and widespread neurodegeneration and subsequent death are observed. Although some biochemical analysis of myelin protein in macular mouse has been reported, detailed histological study of myelination in this mouse model is currently lacking. Since myelin abnormality is one of the neuropathologic findings of human Menkes disease, in this study early myelination in macular mouse brain was evaluated by immunohistochemistry. Two-week-old macular mice and normal littermates were perfused with 4% paraformaldehyde. Immunohistochemical staining of paraffin embedded and vibratome sections was performed using antibodies against either CNPase, cleaved caspase-3 or O4 (marker of immature oligodendrocytes). This staining showed that cerebral myelination in macular mouse was generally hypoplastic and that hypomyelination was remarkable in internal capsule, corpus callosum, and cingulate cortex. In addition, an increased number of cleaved caspase-3 positive cells were observed in corpus callosum and internal capsule. Copper deficiency induced by low copper diet has been reported to induce oligodendrocyte dysfunction and leads to hypomyelination in this mouse model. Taken together, hypomyelination observed in this study in a mouse model of Menkes disease is assumed to be induced by increased apoptosis of immature oligodendrocytes in developing cerebrum, through deficient intracellular copper metabolism.

Histochemistry on vibratome sections of fish tissue: a comparison of fixation and embedding methods

Despite improvements in imaging techniques during recent years, for many non- model systems the fixation of tissues followed by embedding and sectioning for histochemical or immunohistochemical staining remains an important technique in vertebrate histology. The pres- ent study sets out to explore the preservation of histological sections of fish tissues using different preparation techniques. The quality of transverse vibratome sections from trunk segments of the lesser-spotted dogfish Scyliorhinus canicula, Atlantic sturgeon Acipenser oxyrinchus and zebrafish Danio rerio were compared using different fixatives (formaldehyde, paraformaldehyde and zinc-formaldehyde) and embedding methods (gelatine, agarose and low-temperature melting agarose). Our data show that the quality of the vibratome sections for histochemical staining is strongly dependent upon fixation and embedding media. Although paraformaldehyde fixation results in a more pronounced shrinkage of the trunk segment than the other fixatives used , the quality of the sections and the histochemical staining was best with this fixative in zebrafish and dogfish. Additionally, the embedding methods have a strong influence on the quality of the sec- tions. In the dogfish and sturgeon samples, the preferred embedding media were agarose and low-temperature melting agarose, since gelatine often caused shrinkage of the tissues. In conclu- sion, for histochemical examinations, the processing protocols for vibratome sectioning need to be adapted individually to each study organism.

Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining.

BackgroundFor decades, the Vibratome served as a standard laboratory resource for sectioning fresh and fixed tissues. In skilled hands, high quality and consistent fresh unfixed tissue sections can be produced using a Vibratome but the sectioning procedure is extremely time consuming. In this study, we conducted a systematic comparison between the Vibratome and a new approach to section fresh unfixed tissues using a Compresstome. We used a Vibratome and a Compresstome to cut fresh unfixed lymphoid and genital non-human primate tissues then used in situ tetramer staining to label virus-specific CD8 T cells and immunofluorescent counter-staining to label B and T cells. We compared the Vibratome and Compresstome in five different sectioning parameters: speed of cutting, chilling capability, specimen stabilization, size of section, and section/staining quality.ResultsOverall, the Compresstome and Vibratome both produced high quality sections from unfixed spleen, lymph node, vagina, cervix, and uterus, and subsequent immunofluorescent staining was equivalent. The Compresstome however, offered distinct advantages; producing sections approximately 5 times faster than the Vibratome, cutting tissue sections more easily, and allowing production of larger sections.ConclusionsA Compresstome can be used to generate fresh unfixed primate lymph node, spleen, vagina, cervix and uterus sections, and is superior to a Vibratome in cutting these fresh tissues.

Imaging the zebrafish dentition: from traditional approaches to emerging technologies.

The zebrafish, a model organism for which a plethora of molecular and genetic techniques exists, has a lifelong replacing dentition of 22 pharyngeal teeth. This is in contrast to the mouse, which is the key organism in dental research but whose teeth are never replaced. Employing the zebrafish as the main organism to elucidate the mechanisms of continuous tooth replacement, however, poses at least one major problem, related to the fact that all teeth are located deep inside the body. Investigating tooth replacement thus relies on conventional histological methods, which are often laborious, time-consuming and can cause tissue deformations. In this review, we investigate the advantages and limitations of adapting current visualization techniques to dental research in zebrafish. We discuss techniques for fast sectioning, such as vibratome sectioning and high-resolution episcopic microscopy, and methods for in toto visualization, such as Alizarin red staining, micro-computed tomography, and optical projection tomography. Techniques for in vivo imaging, such as two-photon excitation fluorescence and second harmonic generation microscopy, are also covered. Finally, the possibilities of light sheet microscopy are addressed.

Use of labeled tomato lectin for imaging vasculature structures

Intravascular injections of fluorescent or biotinylated tomato lectin were tested to study labeling of vascular elements in laboratory mice. Injections of Lycopersicon esculentum agglutinin (tomato lectin) (50-100 µg/100 µl) were made intravascularly, through the tail vein, through a cannula implanted in the jugular vein, or directly into the left ventricle of the heart. Tissues cut for thin 10- to 12-µm cryostat sections, or thick 50- to 100-µm vibratome sections, were examined using fluorescence microscopy. Tissue labeled by biotinylated lectin was examined by bright field microscopy or electron microscopy after tissue processing for biotin. Intravascular injections of tomato lectin led to labeling of vascular structures in a variety of tissues, including brain, kidney, liver, intestine, spleen, skin, skeletal and cardiac muscle, and experimental tumors. Analyses of fluorescence in serum indicated the lectin was cleared from circulating blood within 2 min. Capillary labeling was apparent in tissues collected from animals within 1 min of intravascular injections, remained robust for about 1 h, and then declined markedly until difficult to detect 12 h after injection. Light microscopic images suggest the lectin bound to the endothelial cells that form capillaries and endothelial cells that line some larger vessels. Electron microscopic studies confirmed the labeling of luminal surfaces of endothelial cells. Vascular labeling by tomato lectin is compatible with a variety of other morphological labeling techniques, including histochemistry and immunocytochemistry, and thus appears to be a sensitive and useful method to reveal vascular patterns in relationship to other aspects of parenchymal development, structure, and function.

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 µm thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 µm is discarded before 200 µm of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 °C. Pieces of outer layer are incubated in 500 µl of Ringer's solution with 2 units of activated papain for 20 min at 37 °C. The reaction is stopped by adding 500 µl 10% fetal calf serum (FCS) in Dulbecco's Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 µl of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.