Vanderbilt University

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology to quantitatively assess infants and children's cortical processing of light touch, speech sounds, and the multisensory processing of the 2 stimuli, without requiring active subject participation or causing discomfort in vulnerable patients.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

The method presented here uses ¹⁸F-Fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 °C) and cold conditions (17 °C) in order to map brown adipose tissue (BAT) in adult human subjects.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

We describe how to implement a battery of behavioral tasks to examine the processing and integration of sensory stimuli in children with ASD. The goal is to characterize individual differences in temporal processing of simple auditory and visual stimuli and relate these to higher order perceptual skills like speech perception.

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The data acquisition procedure for determining embedded sensitivity functions is described. Data is acquired and representative results are shown for a residential scale wind turbine blade.

Here, we present a protocol to fabricate organic thin film solar cells using a mini-slot die coater and related in-line structure characterizations using synchrotron scattering techniques.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of β-cell proliferation. This method is suitable for determining the efficacy of candidate β-cell mitogens.

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications.

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

A 2-azido-1-nitrate-ester can be converted to the corresponding 2-azido-1-trichloroacetimidate in a one-pot procedure. The goal of the manuscript is to demonstrate utility of the microwave reactor in carbohydrate synthesis.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

The purpose of this work is to describe a protocol for creating a practical fat-water phantom that can be customized to produce phantoms with varying fat percentages and volumes.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into naïve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

This study describes a protocol to evaluate the targeting accuracy in the focal plane of an ultrasound-guided high-intensity focused ultrasound phased-array system.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

Dipstick urinalysis is a quick and affordable method of assessing one’s personal state of health. We present a method to perform accurate, low-cost dipstick urinalysis that removes the primary sources of error associated with traditional dip-and-wipe protocols and is simple enough to be performed by lay users at home.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring’s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

A protocol is presented that functionally characterizes mosquito ORs in response to human odors using a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a powerful new technique for exploring the responses of mosquitoes ORs to exposure to human odors.

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans. These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

Spatial distance is a key parameter in assessing hypoxia/reoxygenation injury in a co-culture model of separate endothelial and cardiomyocyte cell layers, suggesting, for the first time, that optimizing the co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of endothelial cells in cardiomyocyte protection.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

The aim of this manuscript is to describe the microsurgical steps required to perform a heterotopic laryngeal transplant in mice. The advantages of this mouse model compared to other animal models of laryngeal transplantation are its cost-effectiveness and the availability of immunologic assays and data.

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Methods For Studying Osteoenergetics And Metabolism

Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs.

Current and Novel Experimental Methods in Ischemia/Reperfusion Research: From Cutting-Edge Models to Innovative Therapies

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

This protocol describes a neonatal porcine model of cardiopulmonary bypass (CPB), with circulatory and cardiac arrest as a tool for studying severe brain damage and other complications secondary to CPB.

Infusing oleic acid continuously into the pulmonary artery of an anesthetized adult pig induces acute respiratory failure, enabling controlled experimentation during acute respiratory decompensation.

The protocol here shows how continuous administration of crystalloids into the central veins of a euvolemic pig/piglet allows for the appropriate investigation of the physiological effects of acute volume overload.

This protocol describes procedures to isolate high-quality nuclei from frozen non-human primate pancreatic islets for use in single-nucleus simultaneous RNA sequencing and ATAC sequencing while preserving the bulk cytosolic fraction for metabolomic analyses from the same samples.

This protocol describes a custom overpressure air system designed to induce closed-system central nervous system (CNS) injuries in mice, including ocular, brain, and spinal cord traumas. The goal of this protocol is to provide a framework for researchers to easily adapt and expand the system for their unique CNS trauma studies.