University of Wisconsin-Madison

In this video, we demonstrate the steps required to run a brain-computer interface experiment, including setting up the EEG cap, calibrating the system, and training the user to move a cursor in two dimensions using imagined movements.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen¹. One measure of CTL quality is their ability to kill specifically². CFSE labeling of target cells can be used to investigate this CTL response quality in vivo^3,4.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary.

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of ˜1,000 cells in peripheral tissues.

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.

Assaying in vitro β-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining β-cell function.

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described. 

Potentiation of the startle reflex is measured via electromyography of the orbicularis oculi muscle during low (uncertain) and high (certain) probability electric shock threat in the Threat Probability Task. This provides an objective measure of distinct negative emotional states (fear/anxiety) for research on psychopathology, substance use/abuse, and broad affective science.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

To replicate laboratory settings, online data collection methods for visual tasks require tight control over stimulus presentation. We outline methods for the use of a web application to collect performance data on two tests of visual attention.

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

The study demonstrates the growth of iridium oxide-reduced graphene oxide (IrO₂-RGO) nanohybrid thin films on irregular and rough screen-printed carbon substrate through a green electrochemical synthesis, and their implementation as a pH sensor with a patterned paper-fluidic platform.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

PI(4,5)P₂ regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P₂ with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P₂ spatial distribution in the plasma membrane at the nanometer scale.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and α/β C-termini, can be quantified.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

This paper presents a protocol for reactive vapor deposition of poly(3,4-ethylenedioxythiophene), poly(3,4-propylenedioxythiophene), and poly(thieno[3,2-b]thiophene) films on glass slides and rough substrates, such as textiles and paper.

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

The goal of this protocol is to evaluate comprehension versus production language learning training through a computer-based experiment in a way that balances inherent task differences between comprehension and production.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

This protocol describes strategies to identify and enrich for cell-state in primary adult mouse neural stem cell cultures by autofluorescence imaging using i) a confocal microscope, ii) a fluorescent activated cell sorter to perform intensity imaging, or iii) a multiphoton microscope to perform fluorescence lifetime imaging.

Early development is dependent on maternally-inherited products, and the role of many of these products is currently unknown. Herein, we described a protocol that uses CRISPR-Cas9 to identify maternal-effect phenotypes in a single generation.

This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus.

Mass spectrometric characterization of neuropeptides provides sequence, quantitation, and localization information. This optimized workflow is not only useful for neuropeptide studies, but also other endogenous peptides. The protocols provided here describe sample preparation, MS acquisition, MS analysis, and database generation of neuropeptides using LC-ESI-MS, MALDI-MS spotting, and MALDI-MS imaging.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

Early life social learning is enhanced by interactions with effectively designed environments. Four events were held at different city parks using inexpensive, temporary waterslides to stimulate social learning. This study describes the prototypes used and the evaluation of the children's interactions.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

The fruit fly (Drosophila melanogaster) is widely used for biological and toxicological research. To expand the utility of flies, we developed an instrument, the serial anesthesia array, that simultaneously exposes multiple fly samples to volatile general anesthetics (VGAs), making it possible to investigate the collateral effects (toxic and protective) of VGAs.

This protocol outlines the steps for utilizing the automated platform Lustro to perform high-throughput characterization of optogenetic systems in yeast.

The rat heterotopic auxiliary liver transplant protocol described here offers a practical investigational tool for exploring mechanisms of hepatic allograft rejection. This model helps to alleviate the surgical hurdles and animal stress of orthotopic liver transplantation in rats.

Here we present a protocol for the measurement of relative telomere length (TL) using the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay. The MMqPCR assay is a repeatable, efficient, and cost-effective method for measuring TL from human DNA in population-based studies.