Vibratome Sections Research Articles

Assessing Different Histological Preparations for Reconstruction of Astrocyte Tridimensional Structure.

Astrocytes are ubiquitous in the brain and spinal cord and display a complex morphology important for the local interactions with neighboring cells, resulting in the modulation of circuit function. Thus, studies focusing on astrocyte physiology in the healthy and diseased brain generally present analyses of astrocytic structure. The labeling method used to visualize the astrocytic structure defines the morphological level to observe and may vary depending on the anatomical sub-regions. The method choice may significantly affect our understanding of their structural diversity. The main goal of this work was to identify a straightforward and efficient protocol for labeling and reconstructing a detailed astrocytic structure to apply and validate in different brain tissue preparations across laboratories. For that, we explored different tissue processing protocols before GFAP labeling to determine the most effective method for reconstructing astrocytic backbones in the mouse hippocampus. Our results show that the reconstruction of astrocytic structure in vibratome sections labeled by free-floating immunofluorescence protocol provides a more practical method to achieve a higher level of detail and arbor complexity in astrocyte backbone reconstruction. Free-floating immunofluorescence labeling is the most reliable method for obtaining better antibody penetration and more detailed astrocyte structure. Finally, we also show that introducing an antigen retrieval step appears useful for visualizing more complete structural details.

The biodistribution of polystyrene nanoparticles administered intravenously in the chicken embryo

Nanoplastics can cause severe malformations in chicken embryos. To improve our understanding of the toxicity of nanoplastics to embryos, we have studied their biodistribution in living chicken embryos. We injected the embryos in the vitelline vein at stages 18–19. We injected polystyrene nanoparticles (PS-NPs) tagged with europium- or fluorescence. Their biodistribution was tracked using inductively-coupled plasma mass spectrometry on tissue lysates, paraffin histology, and vibratome sections analysed by machine learning algorithms. PS-NPs were found at high levels in the heart, liver and kidneys. Furthermore, PS-NPs crossed the endocardium of the heart at sites of epithelial-mesenchymal transformation; they also crossed the liver endothelium. Finally, we detected PS-NPs in the allantoic fluid, consistent with their being excreted by the kidneys. Our study shows the power of the chicken embryo model for analysing the biodistribution of nanoplastics in embryos. Such experiments are difficult or impossible in mammalian embryos. These findings are a major advance in our understanding of the biodistribution and tissue-specific accumulation of PS-NPs in developing animals.

High resolution fluorescence imaging of the alveolar scaffold as a novel tool to assess lung injury

Acute lung injury (ALI) represents an aetiologically diverse form of pulmonary damage. Part of the assessment and diagnosis of ALI depends on skilled observer-based scoring of brightfield microscopy tissue sections. Although this readout is sufficient to determine gross alterations in tissue structure, its categorical scores lack the sensitivity to describe more subtle changes in lung morphology. To generate a more sensitive readout of alveolar perturbation we carried out high resolution immunofluorescence imaging on 200 μm lung vibratome sections from baseline and acutely injured porcine lung tissue, stained with a tomato lectin, Lycopersicon Esculentum Dylight-488. With the ability to resolve individual alveoli along with their inner and outer wall we generated continuous readouts of alveolar wall thickness and circularity. From 212 alveoli traced from 10 baseline lung samples we established normal distributions for alveolar wall thickness (27.37; 95% CI [26.48:28.26]) and circularity (0.8609; 95% CI [0.8482:0.8667]) in healthy tissue. Compared to acutely injured lung tissue baseline tissue exhibited a significantly lower wall thickness (26.86 ± 0.4998 vs 50.55 ± 4.468; p = 0.0003) and higher degree of circularityϕ≤ (0.8783 ± 0.01965 vs 0.4133 ± 0.04366; p < 0.0001). These two components were subsequently combined into a single more sensitive variable, termed the morphological quotient (MQ), which exhibited a significant negative correlation (R2 = 0.9919, p < 0.0001) with the gold standard of observer-based scoring. Through the utilisation of advanced light imaging we show it is possible to generate sensitive continuous datasets describing fundamental morphological changes that arise in acute lung injury. These data represent valuable new analytical tools that can be used to precisely benchmark changes in alveolar morphology both in disease/injury as well as in response to treatment/therapy.

Retrolaminar Demyelination of Structurally Intact Axons in Nonhuman Primate Experimental Glaucoma.

To determine if structurally intact, retrolaminar optic nerve (RON) axons are demyelinated in nonhuman primate (NHP) experimental glaucoma (EG). Unilateral EG NHPs (n = 3) were perfusion fixed, EG and control eyes were enucleated, and foveal Bruch's membrane opening (FoBMO) 30° sectoral axon counts were estimated. Optic nerve heads were trephined; serial vibratome sections (VSs) were imaged and colocalized to a fundus photograph establishing their FoBMO location. The peripheral neural canal region within n = 5 EG versus control eye VS comparisons was targeted for scanning block-face electron microscopic reconstruction (SBEMR) using micro-computed tomographic reconstructions (µCTRs) of each VS. Posterior laminar beams within each µCTR were segmented, allowing a best-fit posterior laminar surface (PLS) to be colocalized into its respective SBEMR. Within each SBEMR, up to 300 axons were randomly traced until they ended (nonintact) or left the block (intact). For each intact axon, myelin onset was identified and myelin onset distance (MOD) was measured relative to the PLS. For each EG versus control SBEMR comparison, survival analyses compared EG and control MOD. MOD calculations were successful in three EG and five control eye SBEMRs. Within each SBEMR comparison, EG versus control eye axon loss was -32.9%, -8.3%, and -15.2% (respectively), and MOD was increased in the EG versus control SBEMR (P < 0.0001 for each EG versus control SBEMR comparison). When data from all three EG eye SBEMRs were compared to all five control eye SBEMRs, MOD was increased within the EG eyes. Structurally intact, RON axons are demyelinated in NHP early to moderate EG. Studies to determine their functional status are indicated.

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging.

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 µm to 70 µm thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

Flat mount preparation for whole-mount fluorescent imaging of zebrafish embryos.

The zebrafish is a widely used model organism for biomedical research due to its ease of maintenance, external fertilization of embryos, rapid embryonic development, and availability of established genetic tools. One notable advantage of using zebrafish is the transparency of the embryos, which enables high-resolution imaging of specific cells, tissues, and structures through the use of transgenic and knock-in lines. However, as the embryo develops, multiple layers of tissue wrap around the lipid-enriched yolk, which can create a challenge to image tissues located deep within the embryo. While various methods are available, such as two-photon imaging, cryosectioning, vibratome sectioning, and micro-surgery, each of these has limitations. In this study, we present a novel deyolking method that allows for high-quality imaging of tissues that are obscured by other tissues and the yolk. Embryos are lightly fixed in 1% PFA to remove the yolk without damaging embryonic tissues and are then refixed in 4% PFA and mounted on custom-made bridged slides. This method offers a simple way to prepare imaging samples that can be subjected to further preparation, such as immunostaining. Furthermore, the bridged slides described in this study can be used for imaging tissue and organ preparations from various model organisms.

Issue Information

Front cover The cellular expression and functions of the sigma-1 receptor (S1R) in the central nervous system (CNS) still remain elusive due to a lack of appropriate tools. For the first time since its discovery over forty years ago, the sigma-1 receptor was specifically detected in all central nervous system (CNS) cell types in vivo by a newly established immunohistochemical protocol. We took advantage of S1R knockout mice to identify a commercial antibody (Ab#61994) specifically recognizing S1Rs in immunoblots. We further established a simple immunohistochemical antigen retrieval method employing 1% SDS to reliably detect S1Rs with Ab#61994 in brain vibratome sections, demonstrating a wide expression of S1R in neurons and all glial cell types. We also generated a new S1R flox mouse for Cre-dependent conditional deletion of S1Rs for functional analysis in vivo. Image content Brain sections from wild-type (WT) and S1R knockout (KO) mice were pre-treated with 1% SDS and immunostained by various S1R antibodies separately. Scale bars = 200 μm. Read the full article ‘Specific detection and deletion of the sigma-1 receptor widely expressed in neurons and glial cells in vivo’ by Q. Liu, Q. Guo, L-P. Fang, H. Yao, A. Scheller, F. Kirchhoff, W. Huang, (J. Neurochem. 2023, vol.164 (6), pp. 764–785) on doi:10.1111/jnc.15693

Spatiotemporal expression pattern of the chicken glucocorticoid receptor during early embryonic development

Glucocorticoids – commonly known as stress hormones - belong to the family of steroid hormones and regulate numerous life essential physiological processes. As lipophilic molecules, glucocorticoids are known to cross the placental barrier in mammals, which – applied for therapeutic reasons or arising from environmental influences – illustrates the role of prenatal stress during embryonic developmental processes. The hormones employ their functions by binding to the glucocorticoid receptor (GR) and thus are involved in regulating the transcription of thousands of genes. Therefore, the aim of this study was to investigate the spatiotemporal expression pattern of the GR during early embryonic vertebrate development, using the chicken embryo as a model organism. The results should contribute to enhance and expand the current understanding of glucocorticoid signaling. By performing in-situ hybridization on whole mount chicken embryos from stage HH10 to HH29 and analyzing vibratome sections of hybridized embryos, we described the spatiotemporal expression pattern of the GR during early embryogenesis. Moreover, we compared the expression pattern of the GR with other developmental markers such as Pax7, Desmin, MyoD and HNK-1 using double in-situ hybridization and immunohistochemistry. We were able to determine the first emergence of GR expression in stage HH13 of chicken development in the cranial area, especially in the muscle anlagen of the branchial arches and of non-somitic neck muscles. Furthermore, we monitored the extension of GR expression pattern throughout later stages and found transcripts of GR during somitogenesis, limb development, myogenesis, neurulation and neural differentiation and moreover during organogenesis of the gastrointestinal organs, the heart, the kidneys and the lungs. Toward later stages, GR expression transitioned from more distinct areas of expression to an increasingly ubiquitous expression pattern. Our results support the notion of an enormous relevance of glucocorticoid signaling during vertebrate embryonic development and contribute to a better understanding of the consequences of prenatal stress and the clinical administration of prenatal glucocorticoids.

Imaging of lactate metabolism in retinal Müller cells with a FRET nanosensor

Müller cells, the glial cells of the retina, provide metabolic support for photoreceptors and inner retinal neurons, and have been proposed as source of the significant lactate production of this tissue. To better understand the role of lactate in retinal metabolism, we expressed a lactate and a glucose nanosensor in organotypic mouse retinal explants cultured for 14 days, and used FRET imaging in acute vibratome sections of the explants to study metabolite flux in real time. Pharmacological manipulation with specific monocarboxylate transporter (MCT) inhibitors and immunohistochemistry revealed the functional expression of MCT1, MCT2 and MCT4 in Müller cells of retinal explants. The introduction of FRET nanosensors to measure key metabolites at the cellular level may contribute to a better understanding of heretofore poorly understood issues in retinal metabolism.

Establishment of a model to assess mumps virus neurovirulence in neonatal Wistar rats

Mumps virus (MuV) is highly neurotropic and neurovirulent, hence, the neurovirulence of virus seeds used in the production of mumps vaccines must be tested. The previous neurovirulence evaluation method involves measuring the area of the cavity in the Lewis neonatal rat brain caused by MuV through paraffin sectioning and hematoxylin-eosin (HE) staining. However, the processes of paraffin sectioning and HE staining are time consuming and complicated. To solve this problem, in this study, a vibratome sectioning system was first deployed to evaluate MuV neurovirulence in the rat brain instead of paraffin sectioning and HE staining. The results showed that the vibratome sectioning method could assess the neurovirulence potential of MuV more objectively and efficiently. In addition, the effects of different MuV doses and the ages of the rats in days on this evaluation method were explored. The results indicate that MuV at no less than 10 50 % cell culture infective dose (CCID50) could cause obvious cavity formation in 1-day-old rat brains. The neonatal rat model developed in this study could evaluate the neurovirulence of different MuV strains with high sensitivity and good repeatability.

Vibratome sectioning of tumors to evaluate the interactions between nanoparticles and the tumor microenvironment ex-vivo.

Nanoparticles have been investigated as drug carriers and promising agents for cancer therapy. However, the tumor microenvironment (TME), which is formed by the tumor, is considered a barrier for nanocarriers to enter the internal tumor tissue. Therefore, the evaluation of the biological distribution of nanocarriers in TME can provide useful information on their role in tumor-targeted drug delivery. Although the tumor-bearing mouse model is commonly used to investigate the distribution of nanocarriers in the TME, there is currently a lack of a testing system to predict the distribution of nanocarriers in tumor tissues, especially in patients. This study revealed that the macrophages and dendritic cells (DCs) were more distributed in the peripheral part than the central part of the tumor, which might be an obstacle to the uniform distribution of nanoparticles in the tumor. In addition, the cellular uptake of gold nanoparticles (AuNR and AuNS) in macrophages and DCs cell lines (RAW264.7 and DC1.2) was markedly different from that in the TME. Hence, the study model of the interaction between nanoparticles and macrophages and DCs has an important impact on the accuracy of the results. The vibratome sections of tumor tissues preserved the spatial distribution of immune cells and tumor cells, and had very little effects on their morphologies and activities. More importantly, we found that the distribution of nanocarriers in vibratome sections was similar to that in tumors in vivo. In all, ex vivo analysis using vibratome sections of tumor tissues provides a more convenient and stable method for elucidating the influences of TME on the distribution of nanocarriers.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to &lt;em&gt;Plasmodiophora brassicae&lt;/em&gt;

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae.

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Single-Neuron Labeling in Fixed Tissue and Targeted Volume Electron Microscopy.

The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 μm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 μm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons.

Confocal Fluorescence Microscopy Investigation for the Existence of Subdomains within Protein Storage Vacuoles in Soybean Cotyledons.

In legumes, the seed storage proteins accumulate within specialized organelles called protein storage vacuoles (PSVs). In several plant species, PSVs are differentiated into subdomains that accumulate different kinds of proteins. Even though the existence of subdomains is common in cereals and legumes, it has not been reported in soybean PSVs. The two most abundant seed proteins of soybean, 7S and 11S globulins, have different temporal accumulation patterns and exhibit considerable solubility differences that could result in differential accretion of these proteins within the PSVs. Here, we employed confocal fluorescent microscopy to examine the presence or absence of subdomains within the soybean PSVs. Eosin-stained sections of FAA-fixed paraffin embedded soybean seeds, when viewed by confocal fluorescence microscopy, revealed the presence of intricate subdomains within the PSVs. However, fluorescence immunolabeling studies demonstrated that the 7S and 11S globulins were evenly distributed within the PSVs and failed to corroborate the existence of subdomains within the PSVs. Similarly, confocal scanning microscopy examination of free-hand, vibratome and cryostat sections also failed to demonstrate the existence of subdomains within PSVs. The subdomains, which were prominently seen in PSVs of FAA-fixed soybean seeds, were not observed when the seeds were fixed either in glutaraldehyde/paraformaldehyde or glutaraldehyde. Our studies demonstrate that the apparent subdomains observed in FAA-fixed seeds may be a fixation artifact.

Confocal Laser and Electron Microscopic Investigation of Gap Junctions in Anaplastic Astrocytomas.

Connexin 43 (Cx43) is a multifunction protein that forms gap junction channels and hemichannels and is suggested to play an essential role in oxygen-glucose deprivation, induced via neuroinflammation during astrocytoma expansion into healthy tissue. To prove this assumption we studied connexin 43 localisation and ultrastructure of gap junctions in samples of malignant brain tumour (anaplastic astrocytomas grade III). For confocal laser microscopy, vibratome sections of tumour fragments were incubated in a mixture of primary antibodies to connexin 43 and glial fibrillary acidic protein (GFAP), then in a mixture of secondary antibodies conjugated with a fluorescent label. After the immunofluorescence study, sections were washed in phosphate buffer, additionally postfixed with 1% OsO4 solution, dehydrated and embedded in epoxy resin by a plane-parallel method. Ultra-thin sections obtained from these samples were contrasted with uranyl acetate and lead citrate and viewed under a Jem 1011 electron microscope. Confocal laser examination detected a positive reaction to Cx43 in the form of point fluorescence. These points were of various sizes. Most of them were localised around or at the intersection of small processes containing GFAP. Electron microscopy of the tumour samples containing the most significant number of Cx43 revealed single and closely spaced gap junctions with a typical ultrastructure on the processes and bodies of tumour cells. Sequential analysis in the fields of view revealed 62 gap junctions in the area of 100μm2. Numerous gap junctions in anaplastic astrocytomas revealed in our study may indicate electrotonic and metabolic transmission between glioma cells, possibly promoting its progression.

Formaldehyde–hardened albumin as a non-penetrating embedding matrix for frozen and vibratome sectioning

In this paper, we describe a protocol for a non-penetrating embedding matrix that can be used for frozen or vibratome sectioning of various formaldehyde-fixed tissue specimens. In our experiments, we wanted to prepare thin frozen sections from miniature specimens for fluorescent staining. As we could not achieve satisfactory results with any of the previously published methods, we have tried to modify the existing protocols, and systematically evaluated the effect of these modifications on the properties of the embedding matrix. The resulting protocol is simple, the matrix gets firmly attached to the tissues, does not cause autofluorescence and enables preparing extremely thin frozen sections. The matrix can be used for 1, embedding miniature specimens from problematic tissues to enable cutting very thin frozen sections, 2, grouping multiple specimens into one large block for simultaneous processing, and 3, dispersing single cells and preparing cell blocks for frozen sectioning.

Brillouin confocal microscopy to determine biomechanical properties of SULEEI-treated bovine pericardium for application in cardiac surgery.

Heart valves are exposed to a highly dynamic environment and underlie high tensile and shear forces during opening and closing. Therefore, analysis of mechanical performance of novel heart valve bioprostheses materials, like SULEEI-treated bovine pericardium, is essential and usually carried out by uniaxial tensile tests. Nevertheless, major drawbacks are the unidirectional strain, which does not reflect the in vivo condition and the deformation of the sample material. An alternative approach for measurement of biomechanical properties is offered by Brillouin confocal microscopy (BCM), a novel, non-invasive and three-dimensional method based on the interaction of light with acoustic waves. BCM is a powerful tool to determine viscoelastic tissue properties and is, for the first time, applied to characterize novel biological graft materials, such as SULEEI-treated bovine pericardium. Therefore, the method has to be validated as a non-invasive alternative to conventional uniaxial tensile tests. Vibratome sections of SULEEI-treated bovine pericardium (decellularized, riboflavin/UV-cross-linked and low-energy electron irradiated) as well as native and GA-fixed controls (n = 3) were analyzed by BCM. In addition, uniaxial tensile tests were performed on equivalent tissue samples and Young's modulus as well as length of toe region were analyzed from stress-strain diagrams. The structure of the extracellular matrix (ECM), especially collagen and elastin, was investigated by multiphoton microscopy (MPM). SULEEI-treated pericardium exhibited a significantly higher Brillouin shift and hence higher tissue stiffness in comparison to native and GA-fixed controls (native: 5.6±0.2 GHz; GA: 5.5±0.1 GHz; SULEEI: 6.3±0.1 GHz; n = 3, p < 0.0001). Similarly, a significantly higher Young's modulus was detected in SULEEI-treated pericardia in comparison to native tissue (native: 30.0±10.4 MPa; GA: 31.8±10.7 MPa; SULEEI: 42.1±7.0 MPa; n = 3, p = 0.027). Native pericardia showed wavy and non-directional collagen fibers as well as thin, linear elastin fibers generating a loose matrix. The fibers of GA-fixed and SULEEI-treated pericardium were aligned in one direction, whereat the SULEEI-sample exhibited a much denser matrix. BCM is an innovative and non-invasive method to analyze elastic properties of novel pericardial graft materials with special mechanical requirements, like heart valve bioprostheses.

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning.

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning