Vibratome Sections Research Articles

Brillouin Spectroscopy as an Innovative Tool to Investigate Biomechanical Properties of Native Human Aortic Valve and Bioprostheses Tissue

Objective: Proper aortic valve (AV) function is enabled by a complex multi-layered extracellular matrix structure and embedded valvular interstitial and endothelial cells (VICs/VECs). In a highly dynamic environment, VICs and VECs respond to biomechanical stimuli thereby influencing valvular matrix remodelling and pathological changes. Thus, it is of cardinal importance to understand the mechanobiology of AV tissue. It is hypothesized that biomechanical properties of native human AV as well as biological AV prostheses tissue can be determined using Brillouin spectroscopy. Methods: Native AV tissue (n=4) and explanted AV prosthesis (n=1) were obtained from operating theatre after AV replacement, 1mm-thick vibratome cuts were prepared (cross section in radial direction) and directly analysed by means of a confocal Brillouin microscope (10x/0.25NA). Here, the inelastically back-scattered laser light (780nm) is analysed by a custom-build Brillouin spectrometer based on a two-stage virtually imaged phased array (VIPA) set-up. Brillouin maps have been acquired pixel-by-pixel (acquisition time 1s/pixel). Tissue slides adjacent to the vibratome sections were fixed and examined histologically. Results: In stenotic AV tissue mineralized regions in the fibrosa exhibit higher Brillouin shift values compared to non-mineralized spongiosa (Figure 1A). The matrix of the examined explanted AV bioprosthesis (glutaraldehyde-fixed porcine AV) showed higher Brillouin shift values than the massive endocarditic vegetation covering both surfaces of the cusp. Calcified regions at the coaptation area were correlated with higher Brillouin shift values (Figure 1B). Conclusions: Brillouin spectroscopy enables detection of differentiated degrees of tissue stiffness within native stenotic and fibrotic human AV. It represents a promising tool to analyze biomechanical properties of human AV. Figure 1. Procedure of Brillouin spectroscopy on (a) stenotic human aortic valve and (b) aortic valve prothesis (from porcine AV cusps) with florid endocarditis and subsequent histological analysis, high Brillouin shift values indicate enhanced stiffness

Examinations of Bilateral Epileptiform Activities in Hippocampal Slices Obtained From Young Mice.

Bilateral interconnections through the hippocampal commissure play important roles in synchronizing or spreading hippocampal seizure activities. Intact hippocampi or bilateral hippocampal slices have been isolated from neonatal or immature rats (6–7 or 12–21 days old, respectively) and the mechanisms underlying the bilateral synchrony of hippocampal epileptiform activities have been investigated. However, the feasibility of examining bilateral epileptiform activities of more developed hippocampal circuitry in vitro remains to be explored. For this, we prepared bilateral hippocampal slices from C57 black mice, a strain commonly used in neuroscience and for genetic/molecular modifications. Young mice (21–24-day-old) were used in most experiments. A 600-μm-thick slice was obtained from each mouse by horizontal vibratome sectioning. Bilateral dorsal hippocampal and connecting dorsal hippocampal commissure (DHC) tissues were preserved in the slice and extrahippocampal tissues were removed. Slices were recorded in a submerged chamber mainly at a room temperature (21–22°C). Bilateral CA3 areas were monitored by extracellular recordings, and unilateral electrical stimulation was used to elicit CA3 synaptic field potentials. The unilateral stimulation could elicit population spikes in the contralateral CA3 area. These contralateral spikes were attenuated by inhibiting synaptic transmission with cobalt-containing medium and were abolished when a cut was made at the DHC. Self-sustained and bilaterally correlated epileptiform potentials were observed following application of 4-aminopyradine and became independent after the DHC cut. Bilateral hippocampal activities were detectable in some slices of adult mice and/or at 35–36°C, but with smaller amplitudes and variable waveforms compared to those observed from slices of young mice and at the room temperature. Together, these observations suggested that examining bilateral epileptiform activities in hippocampal slices of young mice is feasible. The weaknesses and limitations of this preparation and our experimentation are discussed.

Optimized Immunostaining of Embryonic and Early Postnatal Mouse Brain Sections.

The mammalian neocortex, the outer layer of the cerebrum and most recently evolved brain region, is characterized by its unique areal and laminar organization. Distinct cortical layers and areas can be identified by the protein expression of graded transcription factors and molecular determinants that define the identity of different projection neurons. Thus, specific detection and visualization of protein expression is crucial for assessing the identity of neocortical neurons and, more broadly, for understanding early and late developmental mechanisms and function of this complex system. Several immunostaining/immunofluorescence methods exist to detect protein expression. Published protocols vary with regard to subtle details, which may impact the final outcome of the immunofluorescence. Here, we provide a detailed protocol, suitable for both thin cryostat sections and thick vibratome sections, which has successfully worked for a wide range of antibodies directed against key molecular players of neocortical development. Ranging from early technical steps of brains collection down to image analysis and statistics, we include every detail concerning sample inclusion and sectioning, slide storage and optimal antibody dilutions aimed at reducing non-specific background. Routinely used in the lab, our background-optimized immunostaining protocol allows efficient detection of area- and layer- specific molecular determinants of distinct neocortical projection neurons. Graphic abstract: Workflow chart for the optimized immunostaining protocol of mouse brain sections. A. A flow chart for different steps of the optimized immunostaining protocol on both thin cryostat and thick vibratome sections. B. Example for immunostaining against Satb2 and Ctip2 on a thin coronal section (20 μm) at the level of the somatosensory cortex. The first column to the left shows the binning system where 6 bins can be overlaid on the image. On the bottom, an example of counting analysis showing the percentage of marker-positive cells normalized to the total number of DAPI or Hoechst-positive cells. C. Example for immunostaining against Satb2 and Ctip2 on a GFP+ thick vibratome section (200 μm). Images are taken at low magnification (10x, left) and high magnification (40x, right). The graph shows a counting of the percentage of Ctip2-positive neurons normalized to the total number of GFP-electroporated neurons on high-magnification images. Images on B and C are modified from Harb et al. (2016).

TAMI-16. BIOMATERIAL MATRICES TO STUDY GLIOBLASTOMA INVASION

Abstract INTRODUCTION Glioblastoma (GBM) is a highly infiltrative and lethal brain cancer. Previous studies have suggested that GBM tumors are stiffer than healthy brain tissues. We posit that local changes in the mechanical microenvironment near the tumor/tissue interface promote migration of GBM cells away from primary tumors. These studies seek to improve our understanding of how mechanical changes to the microenvironment of GBM tumors and surrounding brain tissue drive GBM progression using an in vitro, 3D model incorporating patient-derived primary GBM cells and a brain matrix-mimetic scaffold in which mechanical modulus can be varied. METHODS GBM xenografts were explanted and vibratome sectioning of fresh tissues for AFM. Hydrogels were fabricated by crosslinking thiolated hyaluronic acid (HA-SH), 8-arm polyethylene glycol (PEG)-norbornene and 4-arm PEG-thiol in the presence of photoinitiator UV light. Mechanical properties were measured using both rheology and AFM. Patient-derived GBM cells were maintained in 3D culture and migration monitored. RESULTS AND DISCUSSION AFM measurements of xenograft and brain tissue suggest that modulus is highest near the tumor center, decreases near the tumor border and is lowest in surrounding brain tissue. Hydrogels were tuned to match in vivo mechanics by altering total thiol content while matrix composition and effective pore size remained constant. GBM cells invaded readily in soft hydrogels, but were not able to migrate in stiff hydrogels. When cultured in platforms including defined regions in which moduli were varied, GBM cells seeded in stiffer regions readily migrated towards softer regions, while cells seeded into a softer matrix did not migrate to stiffer regions. Mechanical properties of the microenvironment appear to have marked effects on tumor cell migration and may drive GBM migration into surrounding tissues. In the long-term, we expect these studies to inform development of new therapeutics targeting GBM tumor invasion through improved understanding of underlying mechanisms.

Ultrastructural GABA immunogold labeling in the substantia nigra pars reticulata of kindled genetic absence epilepsy rats

ABSTRACT Genetic Absence Epilepsy Rats from Strasbourg (GAERS) is a well-known animal model of absence epilepsy and they are resistant to electrical kindling stimulations. The present study aimed to examine possible differences in gamma-aminobutyric acid (GABA) levels and synapse counts in the substantia nigra pars reticulata anterior (SNRa) and posterior (SNRp) regions between GAERS and Wistar rats receiving kindling stimulations. Animals in the kindling group either received six stimulations in the amygdala and had grade 2 seizures or they were kindled, having grade five seizures. Rats were decapitated one hour after the last stimulation. SNR regions were obtained after vibratome sectioning of the brain tissue. GABA immunoreactivity was detected by immunogold method and synapses were counted. Sections were observed by transmission electron microscope and analyzed by Image J program. GABA density in the SNRa region of fully kindled GAERS and Wistar groups increased significantly compared to that of their corresponding grade 2 groups. The number of synapses increased significantly in kindled and grade 2 GAERS groups, compared to kindled and grade 2 Wistar groups, respectively, in the SNRa region. GABA density in the SNRp region of kindled GAERS group increased significantly compared to that of GAERS grade 2 group. In the SNRp region, both kindled and grade 2 GAERS groups were found to have increased number of synapses compared to that of GAERS control group. We concluded that both SNRa and SNRp regions may be important in modulating resistance of GAERS to kindling stimulations.

Solitary chemosensory cells are innervated by trigeminal nerve endings and autoregulated by cholinergic receptors.

Solitary chemosensory cells (SCCs) in the murine nasal epithelium are discrete specialized cells that respond to irritants and activate trigeminal nerve fibers through the release of acetylcholine (ACh), resulting in local neurogenic inflammation. In addition to releasing ACh, SCCs are the exclusive epithelial source of interleukin (IL)-25. In humans, SCCs are significantly expanded in sinonasal polyps (NPs). However, the SCC-trigeminal synapse has yet to be demonstrated in human sinonasal epithelium. Immunofluorescence for trigeminal nerve fiber markers, nicotinic ACh receptors (nChR), and SCC markers was performed in vibratome sections from polyp and healthy turbinate tissue. Quantitative polymerase chain reaction and immunofluorescence of cultured epithelial cells were used to evaluate the expansion of SCCs. Last, intracellular calcium imaging was used to demonstrate cholinergic signaling in sinonasal epithelial cells. Calcitonin gene-related peptide (CGRP) immunostaining was used to identify cholinergic nerve endings, which were only evident in sections from the inferior turbinate and intertwined with SCCs (α-gustducin-positive cells). CGRP-positive nerve endings were not identified in sections from NPs. Human SCCs expressed nChR as well as the ACh synthetic enzyme choline acetyltransferase. Live cell calcium imaging demonstrated functionally active cholinergic signaling in discrete sinonasal epithelial cells, consistent with SCCs. Finally, SCC-specific genes were dramatically upregulated with pretreatment with IL-13 and nicotinic agonists. SCCs are innervated by trigeminal nerve endings in healthy turbinate tissue but not in NPs. SCCs express ACh receptors as well as choline acetyltransferase and, in the setting of a type 2 inflammatory environment, denervated SCCs dramatically expand with nicotinic stimulation.

How to distinguish between different cell lineages sharing common markers using combinations of double in-situ-hybridization and immunostaining in avian embryos: CXCR4-positive mesodermal and neural crest-derived cells

Cell migration plays a crucial role in early embryonic development. The chemokine receptor CXCR4 has been reported to guide migration of neural crest cells (NCCs) to form the dorsal root ganglia (DRG) and sympathetic ganglia (SG). CXCR4 also plays an important part during the formation of limb and cloacal muscles. NCCs migration and muscle formation during embryonic development are usually considered separately, although both cell lineages migrate in close neighbourhood and have markers in common. In this study, we present a new method for the simultaneous detection of CXCR4, mesodermal markers and NCCs markers during chicken embryo developmental stages HH18–HH25 by combining double whole-mount in situ hybridization (ISH) and immunostaining on floating vibratome sections. The simultaneous detection of CXCR4 and markers for the mesodermal and neural crest cells in multiple labelling allowed us to compare complex gene expression patterns and it could be easily used for a wide range of gene expression pattern analyses of other chicken embryonic tissues. All steps of the procedure, including the preparation of probes and embryos, prehybridization, hybridization, visualization of the double labelled transcripts and immunostaining, are described in detail.

Gelatin embedding and LED autofluorescence reduction for rodent spinal cord histology

We present two innovations in histological technique for rodent spinal cord: gelatin embedding and LED photobleaching. Gelatin embedding uses liquid gelatin solution to permeate delicate biological structures then solidify to provide mechanical support throughout dissection, vibratome sectioning, and staining. LED photobleaching uses high-intensity visible light during blocking and primary incubations to reduce autofluorescence in tissue sections before fluorescent secondaries are added. We found gelatin embedding improved mechanical stability without interfering with immunohistochemical staining. Gelatin embedding also preserved some spinal roots and provided an opportunity for dye-less and cut-less tracking of left/right orientation during free-floating staining, which is valuable for tissue samples that have no spare areas that can be marked. LED photobleaching greatly reduced autofluorescence and added essentially no extra time or labor to the process. Descriptions of the techniques and characterization data are provided.

Vibratome Sectioning and Clearing for Easing Studies of Cassava Embryo Formation.

This work describes the application of clearing on vibratome sections to study the embryo formation in cassava. This procedure provides high-resolution images and reduces significantly the number of sections that need to be analyzed per ovule. This methodology was instrumental for the development of the protocol for embryo rescue in cassava. It has been also applied to monitor the embryo formation response when optimizing seed setting from regular and broad crosses for cassava breeding. Broad crosses between cassava and castor bean (incompatible-euphorbiaceae species) were made aiming to induce doubled haploids through the elimination of the incompatible-male parent genome as done in cereals. Castor bean is widely available and provides continues supply of pollen. Our results suggest that this methodology is easy and effective to assess the response of hundreds of cassava ovules pollinated with castor bean pollen, allowing the identification of multicellular structures in the embryo sac without apparent formation of endosperm. The protocol is also useful when developing and optimizing a methodology to induce doubled haploids in cassava via gynogenesis or from ovules pollinated with irradiated cassava pollen.

From 2D to 3D: Promising Advances in Imaging Lung Structure.

The delicate structure of murine lungs poses many challenges for acquiring high-quality images that truly represent the living lung. Here, we describe several optimized procedures for obtaining and imaging murine lung tissue. Compared to traditional paraffin cross-section and optimal cutting temperature (OCT), agarose-inflated vibratome sections (aka precision-cut lung slices), combines comparable structural preservation with experimental flexibility. In particular, we discuss an optimized procedure to precision-cut lung slices that can be used to visualize three-dimensional cell-cell interactions beyond the limitations of two-dimensional imaging. Super-resolution microscopy can then be used to reveal the fine structure of lung tissue's cellular bodies and processes that regular confocal cannot. Lastly, we evaluate the entire lung vasculature with clearing technology that allows imaging of the entire volume of the lung without sectioning. In this manuscript, we combine the above procedures to create a novel and evolutionary method to study cell behavior ex vivo, trace and reconstruct pulmonary vasculature, address fundamental questions relevant to a wide variety of vascular disorders, and perceive implications to better imaging clinical tissue.

Sex-specific features of spine densities in the hippocampus

Previously, we found that in dissociated hippocampal cultures the proportion of large spines (head diameter ≥ 0.6 μm) was larger in cultures from female than from male animals. In order to rule out that this result is an in vitro phenomenon, we analyzed the density of large spines in fixed hippocampal vibratome sections of Thy1-GFP mice, in which GFP is expressed only in subpopulations of neurons. We compared spine numbers of the four estrus cycle stages in females with those of male mice. Remarkably, total spine numbers did not vary during the estrus cycle, while estrus cyclicity was evident regarding the number of large spines and was highest during diestrus, when estradiol levels start to rise. The average total spine number in females was identical with the spine number in male animals. The density of large spines, however, was significantly lower in male than in female animals in each stage of the estrus cycle. Interestingly, the number of spine apparatuses, a typical feature of large spines, did not differ between the sexes. Accordingly, NMDA-R1 and NMDA-R2A/B expression were lower in the hippocampus and in postsynaptic density fractions of adult male animals than in those of female animals. This difference could already be observed at birth for NMDA-R1, but not for NMDA-R2A/B expression. In dissociated embryonic hippocampal cultures, no difference was seen after 21 days in culture, while the difference was evident in postnatal cultures. Our data indicate that hippocampal neurons are differentiated in a sex-dependent manner, this differentiation being likely to develop during the perinatal period.

Vibratome Sectioning of Plant Materials for (Immuno)cytochemical Staining.

A vibrating microtome is widely used to produce good-quality sections of plant organs or tissues. This method allows for an improved preservation of antigenicity and structure and is compatible with most (immuno)cytochemical staining procedures.

Hybridization Chain Reaction for Quantitative and Multiplex Imaging of Gene Expression in Amphioxus Embryos and Adult Tissues.

In situ hybridization (ISH) methods remain the most popular approach for profiling the expression of a gene at high spatial resolution and have been broadly used to address many biological questions. One compelling application is in the field of evo-devo, where comparing gene expression patterns has offered insight into how vertebrate development has evolved. Gene expression profiling in the invertebrate chordate amphioxus (cephalochordate) has been particularly instrumental in this context: its key phylogenetic position as sister group to all other chordates makes it an ideal model system to compare with vertebrates and for reconstructing the ancestral condition of our phylum. However, while ISH methods have been developed extensively in vertebrate model systems to fluorescently detect the expression of multiple genes simultaneously at a cellular and subcellular resolution, amphioxus gene expression profiling is still based on single-gene nonfluorescent chromogenic methods, whose spatial resolution is often compromised by diffusion of the chromogenic product. This represents a serious limitation for reconciling gene expression dynamics between amphioxus and vertebrates and for molecularly identifying cell types, defined by their combinatorial code of gene expression, that may have played pivotal roles in evolutionary innovation. Herein we overcome these problems by describing a new protocol for application of the third-generation hybridization chain reaction (HCR) to the amphioxus, which permits fluorescent, multiplex, and quantitative detection of gene expression in situ, within the changing morphology of the developing embryo, and in adult tissues. A detailed protocol is herein provided for whole-mount preparations of embryos and vibratome sections of adult tissues.

Basic Neuroanatomical Methods.

This unit covers some basic procedures that are common to a wide range of neuroanatomical protocols for brain tissue. Procedures are provided for preparation of unfixed fresh brain tissue as well as for perfusion fixation of animals to obtain fixed neural tissue. A variety of methods for sectioning are described, including frozen sectioning using a cryostat or microtome and sectioning with a vibratome. The choice of sectioning method depends on how the brain has been prepared and what histochemical method is to be used. A fluorescent immunohistochemical method to localize endogenous molecules as well as induced markers such as green fluorescent protein and red fluorescent protein is also provided. Additionally, three post-sectioning procedures are described: defatting of slide-mounted sections, fluorescent Nissl staining, and thionin staining of sections. Finally, support protocols are provided, describing a method for maintaining the correct order of cut tissue, whether rostral to caudal or lateral to medial; a procedure for subbing slides with gelatin, which is necessary in some protocols in order for sections to adhere to slides; and preparation of custom 3D-printed 10- or 20-well tissue plates and trays for subsequent immunostaining. Published 2019. U.S. Government. Basic Protocol 1: Preparation of unfixed fresh-frozen brain tissue Basic Protocol 2: Perfusion fixation Basic Protocol 3: Cryostat sectioning of frozen brain tissue Basic Protocol 4: Sliding-microtome sectioning of fixed brain tissue Basic Protocol 5: Vibratome and Compresstome sectioning Support Protocol 1: Tissue collection in a 1-in-10 series Support Protocol 2: Preparation of gelatin-subbed microscope slides Support Protocol 3: Custom 3D-printed 10- and 20-well tissue plates Basic Protocol 6: Post-sectioning procedures I: Fluorescent immunohistochemical localization Basic Protocol 7: Post-sectioning procedures II: Defatting Basic Protocol 8: Post-sectioning procedures III: Nissl staining Basic Protocol 9: Post-sectioning procedures IV: Thionin staining.

Methods in Brain Development of Molluscs.

Representatives of the phylum Mollusca have long been important models in neurobiological research. Recently, the routine application of immunocytochemistry and gene expression analyses in combination with confocal laserscanning microscopy has allowed fast generation of highly detailed reconstructions of neural structures of even the smallest multicellular animals, including early developmental stages. As a consequence, large-scale comparative analyses of neurogenesis-an important prerequisite for inferences concerning the evolution of animal nervous systems-are now possible in a reasonable amount of time. Herein, we describe immunocytochemical staining and in situ hybridization protocols for both, whole-mount preparations of developmental stages-usually 70-300μm in size-as well as for vibratome and cryostat sections of complex brains. Although our procedures have been optimized for marine molluscs, they may easily be adapted to other (marine) organisms by the creative neurobiologist.

Step by Step Golgi-Cox Staining for Cryosection.

Golgi staining, though invented hundreds of years ago, is still a reliable method to study the cytoarchitecture of the brain. Almost all published Golgi staining protocols and methods were used for microtome, and rarely applied in cryosection, which restricted the application of this technique. Currently, several commercial Golgi-stain kits are available for both vibratome section and cryosection, but these kits are costly, and it is still challenging for researchers to obtain significant results. In the present study, we described a protocol of Golgi-Cox Staining for Cryosection, with the modified cryosection protection solution based on the Golgi-Cox method, which makes cryosection easy to apply in the section of the Golgi-Cox impregnated tissue. Our methods provide a low-cost and simple option for Golgi staining, and it will facilitate researchers to obtain useful Golgi staining results for neuronal architecture studies.

Beyond the whole-mount phenotype: high-resolution imaging in fluorescence-based applications on zebrafish

ABSTRACTZebrafish is now widely used in biomedical research as a model for human diseases, but the relevance of the model depends on a rigorous analysis of the phenotypes obtained. Many zebrafish disease models, experimental techniques and manipulations take advantage of fluorescent reporter molecules. However, phenotypic analysis often does not go beyond establishing overall distribution patterns of the fluorophore in whole-mount embryos or using vibratome or paraffin sections with poor preservation of tissue architecture and limited resolution. Obtaining high-resolution data of fluorescent signals at the cellular level from internal structures mostly depends on the availability of expensive imaging technology. Here, we propose a new and easily applicable protocol for embedding and sectioning of zebrafish embryos using in-house prepared glycol methacrylate (GMA) plastic that is suited for preservation of fluorescent signals (including photoactivatable fluorophores) without the need for antibodies. Four main approaches are described, all involving imaging fluorescent signals on semithin (3 µm or less) sections. These include sectioning transgenic animals, whole-mount immunostained embryos, cell tracking, as well as on-section enzyme histochemistry.

An Unbiased Approach of Sampling TEM Sections in Neuroscience.

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

Strain-Related Differences in Mouse Neonatal Hypoxia-Ischemia

Neonatal hypoxic-ischemic brain injury is commonly studied by means of the Vannucci procedure in mice or rats (unilateral common carotid artery occlusion followed by hypoxia). Previously, we modified the postnatal day 7 (P7) rat procedure for use in mice, and later demonstrated that genetic strain strongly influences the degree of brain injury in the P7 mouse model of hypoxia-ischemia (HI). Recently, the P9 or P10 mouse brain was recognized as the developmental equivalent of a term neonatal human brain, rather than P7. Consequently, the Vannucci procedure has again been modified, and a commonly used protocol employs 10% oxygen for 50 min in C57Bl/6 mice. Strain differences have yet to be described for the P9/P10 mouse model. In order to determine if the strain differences we previously reported in the P7 mouse model are present in the P9 model, we compared 2 commonly used strains, CD1 and C57Bl/6J, in both the P7 (carotid ligation [in this case, right] followed by exposure to 8% oxygen for 30 min) and P9 (carotid ligation [in this case left] followed by exposure to 10% oxygen) models of HI. Experiments using the P7 model were performed in 2001–2012 and those using the P9 model were performed in 2012–2016. Five to seven days after the HI procedure, mice were perfused with 4% paraformaldehyde, their brains were sectioned on a Vibratome (50 µm) and alternate sections were stained with Perl’s iron stain or cresyl violet. Brain sections were examined microscopically and scored for the degree of injury. Since brains in the P7 group had been scored previously with a slightly different system, they were reanalyzed using our current scoring system which scores injury in 11 regions: the anterior, middle, and posterior cortex; the anterior, middle, and posterior striatum; CA1, CA2, CA3, and the dentate gyrus of the hippocampus and thalamus, on a scale from 0 (none) to 3 (cystic infarct) for a total score of 0–33. Brains in the P9 group were scored with the same system. Given the same insult, the P7 CD1 mice had greater injury than the C57Bl/6J mice, which agrees with our previous findings. The P9 CD1 mice also had greater injury than the C57Bl/6J mice. This study confirms that CD1 mice are more susceptible to injury than C57Bl/6J mice and that strain selection is important when using mouse models of HI.

Organotypic Brain Slice Cultures

Ex vivo cell culture models are of particular interest for neurobiologists, as these allow the study of brain cells in an isolated environment. Organotypic brain slice cultures allow growth of three-dimensional brain tissue that conserves the cellular architecture. This unit describes the preparation and culturing of organotypic brain slices from mice. In three basic protocols, the general procedure, the specific preparation of chopper slices, and slicing of whole-brain vibratome sections are described. Support protocols explain the use of postnatal or adult mice, the preparation of coronal or sagittal slices, the preparation of co-cultures, post-processing of slices, the use of extra filter membranes, and the re-use of membrane inserts. This unit provides an easy-to-understand methodological introduction to brain slice cultures. © 2018 by John Wiley & Sons, Inc.