Harvard Medical School

Monitoring Actin Disassembly with Time-lapse Microscopy

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Studying Aggression in Drosophila (fruit flies)

Interview with Edward Kravitz

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

Denaturing Gradient Gel Electrophoresis (DGGE)

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Biology of Microbial Communities - Interview

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

Title Cell Encapsulation by Droplets

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block^[1].

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children.

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Here we show how to do retro-orbital injection in adult zebrafish.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

This video demonstrates how to encapsulate and culture cancer cells in PuraMatrix, a commercially available self assembling peptide gel.

This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.

Phosphenes are transient percepts of light that can be induced by applying Transcranial Magnetic Stimulation (TMS) to visually sensitive regions of cortex. We demonstrate a standard protocol for determining the phosphene threshold value and introduce a novel method for quantifying and analyzing perceived phosphenes.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability^1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation ^3-5.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via β-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

High-definition transcranial direct current stimulation (HD-tDCS), with its 4x1-ring montage, is a noninvasive brain stimulation technique that combines both the neuromodulatory effects of conventional tDCS with increased focality. This article provides a systematic demonstration of the use of 4x1 HD-tDCS, and the considerations needed for safe and effective stimulation.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3^rd order mesenteric arteries in c57 and sGCα₁^-/- mice using pressure myography.

Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that has shown initial therapeutic effects in several neurological conditions. The main mechanism underlying these therapeutic effects is the modulation of cortical excitability. Therefore, online monitoring of cortical excitability would help guide stimulation parameters and optimize its therapeutic effects. In the present article we review the use of a novel device that combines simultaneous tDCS and EEG monitoring in real time.

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease. An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates and analyzed for a variety of biomarkers.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Cerebral perfusion is maintained across a range of pressures via cerebral autoregulation. However, characterizing autoregulation requires prominent pressure fluctuations at regulated frequencies. The described protocol will show how oscillatory lower body negative pressure can generate pressure fluctuations to provide data for projection pursuit regression for quantification of the autoregulatory curve.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Nanopodia are thin but fragile membrane channels that extend up to 100 μm from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 °C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

The protocol describes the corneal micropocket assay as developed in mice.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is <30 min compared to 60 - 100 min using alternative methods.

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

Patients implanted with intracranial electrodes provide a unique opportunity to record neurological data from multiple areas of the brain while the patient performs behavioral tasks. Here, we present a method of recording from implanted patients that can be reproducible at other institutions with access to this patient population.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Vascularized composite allotransplantation (VCA) offers unparalleled restoration of function and form following devastating musculoskeletal and soft tissue injury. Surgical techniques have developed rapidly, but immunological and functional outcomes require further study. We described a rigorous non-human primate model of orthotopic upper extremity transplantation for pre-clinical studies immediately preceding clinical translation.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 °C).

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel’s cartilage.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice.  

During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong "loser" effects.

Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the naïve-primed pluripotent transition.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

This study investigated lower-limb kinematics and ground reaction force (GRF) during moderate high-heeled jogging and running. Subjects were divided into groups of experienced wearers and inexperienced wearers. A three-dimensional motion analysis system with a configured force platform captured lower-limb joint movements and GRF.

The goal of this protocol is to outline the collection and processing of human surgical samples for multiple downstream applications in vestibular schwannoma and Schwann cell research.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

We describe a method for the stereotactically-guided location, exposure, and ablation of the auditory cortex in rats. The localization of the ablation is assessed using a coordinate map postmortem.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

This study provides an updated home-based tDCS protocol that enables subjects to receive the beneficial effects of tDCS at home with an easy to use device with settings to control the use and dosage, enhancing the feasibility for long-term use at home.

In this protocol, we describe how to make Loewe additivity-based drug interaction measurements for pairwise and three-way drug combinations.

This study aims to develop a standard protocol of intra-operative neural monitoring of thyroid surgery in a porcine model. Here, we present a protocol to demonstrate general anesthesia, to compare different types of electrodes, and to investigate the electrophysiological characteristics of the normal and injured recurrent laryngeal nerves.

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

Here, we present a protocol on the use of intraoperative ultrasound in spinal surgery, particularly in cases of intradural lesions and lesions in the ventral spinal canal when using a posterior approach.

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (T_HGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of T_HGM, and analysis of differentiated T_HGM cells. This method can be applied to studies of the regulation and function of T_HGM cells.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using ¹⁸F-FDG PET/CT.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer's Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

The combined use of transcranial direct current stimulation and robotic therapy as an add-on for conventional rehabilitation therapy may result in improved therapeutic outcomes due to modulation of brain plasticity. In this article, we describe the combined methods used in our institute for improving motor performance after stroke.

This manuscript describes the synthesis of a single-wall carbon nanotube (SWCNT)-conjugated MALAT1 antisense gapmer DNA oligonucleotide (SWCNT-anti-MALAT1), which demonstrates the reliable delivery of the SWCNT and the potent therapeutic effect of anti-MALAT1 in vitro and in vivo. Methods used for synthesis, modification, conjugation, and injection of SWCNT-anti-MALAT1 are described.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

We present a novel combined behavioral and neuroimaging protocol employing real-time video projection for the purpose of characterizing the neural correlates associated with mirror therapy within the magnetic resonance imaging scanner environment in leg amputee subjects with phantom limb pain.

Here, we present a protocol to evaluate the differences in injury mechanisms between professional and amateur players when performing a badminton maximal right lunge movement by analyzing lower limb kinematics.

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

We performed functional MRI using a novel MRI-compatible hand-induced robotic device to evaluate its utility for monitoring hand motor function in individuals recovering from neurological deficits.

Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change.

We describe a strategy for how to use RNA samples from unreferenced Pacific oyster specimens, and evaluate the genetic material by comparison with publicly available genome data to generate a virtually sequenced cDNA library.

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

Brush cells are rare cholinergic chemosensory epithelial cells found in the naïve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 Isl^MN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

This manuscript describes an ice-free cryopreservation method for large quantities of rat hepatocytes whereby primary cells are pre-incubated with cryoprotective agents at a low concentration and vitrified in large droplets.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Little Fish, Big Questions: A Collection of Modern Techniques for Mexican Tetra Research

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers’ method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

This simple and highly adaptable system device for the inhalation of high-concentration nitric oxide (NO) gas does not require mechanical ventilators, positive pressure, or high gas flows. Standard medical consumables and a snug-fitting mask are used to safely deliver NO gas to spontaneously breathing subjects.

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR.

This protocol, which was originally reported by Fernandez-Godino et al. in 2016¹, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

This study presents a highly reproducible large animal model of renal ischemia-reperfusion injury in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries for 60 min and reperfusion for 24 h.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

The single-position, prone, lateral approach allows for both lateral lumbar interbody placement and direct posterior decompression with pedicle screw placement in one position.

This protocol provides experimental tools to evaluate macropinocytic uptake of nutrients (carbohydrate and protein) by mTORC1-hyperactive cells. Detailed steps to quantify the uptake of fluorescently labeled dextran and bovine serum albumin (BSA) are described.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

This article describes how to effectively utilize three cryo-EM processing platforms, i.e., cryoSPARC v3, RELION-3, and Scipion 3, to create a single and robust workflow applicable to a variety of single-particle data sets for high-resolution structure determination.

The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research.

The safe application of newly developed surgical energy devices in thyroid/parathyroid surgery attracts the attention of surgeons. Animal experimental models can avoid unnecessary trials and errors in human surgery. This report aims to demonstrate electrophysiological and thermographic methods to evaluate the safety parameters of SEDs in thyroid/parathyroid surgery.

The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening.