The authors are deeply indebted to the efforts of Hughes lab members Drs Seetharamaiah Attili, Jana Koth, Fernanda Bajanca, Victoria C. Williams, Yaniv Hinits, Giorgia Bergamin, and Vladimir Snetkov for development of the described protocols, and to Henry Roehl, Christina Hammond, David Langenau and Peter Currie for sharing plasmids or zebrafish lines. SMH is a Medical Research Council (MRC) Scientist with Programme Grant G1001029, MR/N021231/1, and MR/W001381/1support. MA held a MRC Doctoral Training Programme PhD Studentship&#160;from King&#39;s College London. This work benefited from the trigonometrical input of David M. Robinson, scholar, mentor, and friend.

The authors declare no competing interests.

Here we report a method for accurate and efficient estimation of the muscle volume of live zebrafish larvae at stages or in genetic variants in which pigmentation is not a big hinderance to imaging and when transient anesthesia and/or immobilization is well tolerated. Whereas we have employed laser scanning confocal microscopy, the approaches described are applicable to spinning disk confocal or light sheet microscopy and to any other method that creates stacks of images at distinct focal planes. A series of increasingly...

Regulated tissue growth, comprising increase in cell number (hyperplasia) and/or cell size (hypertrophy), is a crucial factor in development, regeneration, and ecological and evolutionary adaptation. Despite huge advances in molecular genetic understanding of both cell and developmental biology over recent decades, mechanistic understanding of the regulation of tissue and organ size is still in its infancy. One reason for this lacuna in knowledge is the difficulty of quantifying tissue growth in living organisms with the necessary spatial and temporal accuracy.
Various aspects of growth of whole organisms can be measured repeatedly over time, revealing growth curves for each individual1,2,3,4,5. Increasingly sophisticated scanning methods, such as dual X-ray absorptiometry (DXA), computerized tomography (CT), and magnetic resonance imaging (MRI), permit the tracking of growth of whole organs and other body regions (for example, individual identified skeletal muscles) in single individuals, both human and in model organisms6,7,8,9,10. However, these methods do not yet have the resolution to reveal individual cells and thus the links between cellular behaviors and tissue level growth have been hard to discern. To make such links, traditional studies have often relied upon cohorts of similar individual animals, a few of which are sacrificed at successive timepoints and then analyzed in cytological detail. Such approaches require averaging the observed changes across groups of (preferably similar, but nevertheless variable) individuals and thus suffer from a lack of temporal and spatial resolution, making it hard to find correlated events at the cellular level suggestive of cause and effect.
Studies on invertebrate model organisms, initially in C. elegans and D. melanogaster, have circumvented these problems by developing optical microscopy to achieve cellular resolution and accurately measure growth over time in single individuals. Such studies have revealed strikingly invariant cell lineage behaviors in the growth of these small model organisms11,12,13,14,15,16,17. However, many animals, including all vertebrates, have indeterminate cell lineages, and control tissue growth by mysterious feedback processes that serve to turn the genetically-encoded growth program into a functional three dimensional organism with all its constituent tissues and organs suitably matched in size. To understand these complex growth processes, it is desirable to image whole tissues or organs over time in single individuals that can be experimentally manipulated by genetic, pharmacological or other interventions at a time of choice and the effect subsequently analyzed.
Each vertebrate skeletal muscle has a defined size, shape and function, and well-characterized interactions with adjacent tissues, such as bone, tendon, and nerves. Some muscles are small, lie just under the skin and are therefore good candidates for high-resolution imaging studies. Similar to most organs, each muscle grows throughout embryonic, postnatal, and juvenile life, before reaching a stable adult size. Muscle, however, also has a unique ability to change size during adult life, dependent upon use and nutrition18, and this property has a major impact on organismal fitness, sporting performance, and independent living. Loss of muscle mass and function in old age, sarcopenia, is an issue of increasing concern for societies faced with an ageing population19,20,21.
We and others have focused on the growth of defined blocks of skeletal muscle tissue in the segmentally-repeating body of zebrafish larvae, as an apparently closed system containing several hundred cells in which tissue growth, maintenance, and repair can be observed and manipulated22,23,24,25,26. While some quantitative work has previously been reported25,26,27,28,29,30,31,32,33,34,35, no detailed and validated method of measuring muscle growth in cellular detail in individual vertebrate organisms over time is available. Here an efficient protocol for how to perform such repeated measurements is described, along with validation, and an example of its use to analyze changes in both hypertrophic and hyperplastic growth in response to altered electrical activity is provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesive, Blu Tack</td>
			<td>Bostik</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Aerosol vacuum&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Agarose, low gelling temperature</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td>Once melted, keep at 37oC in a block heater to remain in liquid form for repeated use.</td>
		</tr>
		<tr>
			<td>Block heater</td>
			<td>Cole-Parmer</td>
			<td>SBH130</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BODIPY FL C5-ceramide</td>
			<td>Thermo Scientific</td>
			<td>D3521</td>
			<td>To be diluted in fish water and used at 5 &#181;M for overnight incubation.</td>
		</tr>
		<tr>
			<td>Crocodile clips and wires</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fiji/imageJ</td>
			<td>National Institutes of Health, NIH</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fish medium, Fish water</td>
			<td>-</td>
			<td>-</td>
			<td>Circulating system water collected from the fish facility.</td>
		</tr>
		<tr>
			<td>Fish medium, E3 medium</td>
			<td>-</td>
			<td>-</td>
			<td>E3 is described in The Zebrafish Book. http://zfin.org (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4 in distilled water).</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Leica</td>
			<td>Leica MZ16F</td>
			<td>Fluorescence microscope of other kind are also expected to be suitable.</td>
		</tr>
		<tr>
			<td>Glass needle</td>
			<td>World Precision Instruments, Inc.</td>
			<td>1B100-6</td>
			<td>To be fire-polished to prevent damage of the embryos during manipulation.</td>
		</tr>
		<tr>
			<td>Grass stimulator</td>
			<td>Grass Instruments</td>
			<td>S88</td>
			<td>Stimulators of other kind are also expected to be suitable.</td>
		</tr>
		<tr>
			<td>Kimwipes, Delicate Task Wipers</td>
			<td>Kimberly-Clark Professional</td>
			<td>13258179</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Laser scanning microscope (LSM)&#160;</td>
			<td>Zeiss</td>
			<td>Zeiss LSM 5 Exciter 
			Zeiss LSM 880</td>
			<td>LSM of other kind are also expected to be suitable.</td>
		</tr>
		<tr>
			<td>Nunc Cell-Culture Treated, 6-well plate</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Objective, 20&#215;/1.0W water immersion</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pasteur Pipette, Graduated 1 mL</td>
			<td>Starlab Group</td>
			<td>E1414-0100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pasteur Pipette, Micro Fine Tip 1 mL</td>
			<td>Starlab Group</td>
			<td>E1414-1100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Petri dish, 60 mm</td>
			<td>Sigma-Aldrich</td>
			<td>P5481</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Plasmid, CMV-Cerulean</td>
			<td>Christina L. Hammond (University of Bristol)</td>
			<td></td>
			<td>pCS2+_cerulean_kanR plasmid injected at 25-75 pg at one-cell stage.&#160; Citation: Bussman J, and Schulte-Merker S. (2011) Development 138:4327-4332. doi: 10.1242/dev.068080.</td>
		</tr>
		<tr>
			<td>Plasmid, pCS-mCherry-CAAX</td>
			<td>Henry Roehl (University of Sheffield)</td>
			<td>-</td>
			<td>For in vitro transcription using the SP6 promoter (plasmids containing other membrane labelling markers can be used); 
			synthesised capped mRNA to be injected at 100-200 pg at one-cell stage.</td>
		</tr>
		<tr>
			<td>Pulse Controller&#160;</td>
			<td>Hoefer Scientific Instruments</td>
			<td>PC750</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Soldering iron</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>Ethyl 3-aminobenzoate methanesulfonate/ MS-222; to be dissolved in fish water and used at 0.6 mM.</td>
		</tr>
		<tr>
			<td>Volocity</td>
			<td>Perkin Elmer/Quorum Technologies Inc</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Watchmaker forceps, No. 5</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Wire, Platinum</td>
			<td>Goodfellow</td>
			<td>PT005142/12</td>
			<td>0.40 mm in diameter; an expensive alternative of silver.</td>
		</tr>
		<tr>
			<td>Wire, Silver</td>
			<td>Acros Organics</td>
			<td>317730010</td>
			<td>0.25 mm in diameter (a range of diameter i.e. 0.25-0.5 mm had been tested, which produced similar results).</td>
		</tr>
		<tr>
			<td>Zebrafish, myog:H2B-mRFP</td>
			<td>David M. Langenau (Massachusetts General Hospital; Harvard Stem Cell Institute)</td>
			<td>-</td>
			<td>ZFIN official name: Tg(myog:Hsa.HIST1H2BJ-mRFP), fb121Tg.&#160; http://zfin.org/ZDB-ALT-160803-2&#160; Citation: Tang Q, Moore JC, Ignatius MS, Tenente IM, Hayes MN, Garcia EG, Torres Yord&#225;n N, Bourque C, He S, Blackburn JS, Look AT, Houvras Y, Langenau DM. Imaging tumour cell heterogeneity following cell transplantation into optically clear immune-deficient zebrafish. Nat Commun. 2016 Jan 21;7:10358. doi: 10.1038/ncomms10358.</td>
		</tr>
		<tr>
			<td>Zebrafish, &#945;-actin:mCherry-CAAX</td>
			<td>Peter D. Currrie (ARMI, Monash University)</td>
			<td>-</td>
			<td>ZFIN official name: Tg(actc1b:mCherry-CAAX), pc22Tg.&#160; http://zfin.org/ZDB-ALT-150224-2 Citation: Berger J, Tarakci H, Berger S, Li M, Hall TE, Arner A, and Currie PD. Loss of Tropomodulin4 in the zebrafish mutant tr&#228;ge causes cytoplasmic rod formation and muscle weakness reminiscent of nemaline myopathy. Dis Model Mech. 2014 Dec;7(12):1407-15. doi: 10.1242/dmm.017376.</td>
		</tr>
		<tr>
			<td>Zebrafish, &#946;-actin:HRAS-EGFP</td>
			<td>-</td>
			<td>-</td>
			<td>ZFIN official name: Tg(Ola.Actb:Hsa.HRAS-EGFP), vu119Tg. http://zfin.org/ZDB-ALT-061107-2&#160; Citation: Cooper MS, Szeto DP, Sommers-Herivel G, Topczewski J, Solnica-Krezel L, Kang HC, Johnson I, and Kimelman D. Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP. Dev Dyn. 2005 Feb;232(2):359-68. doi: 10.1002/dvdy.20252.</td>
		</tr>
		<tr>
			<td>ZEN software</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

real time and repeated measurement of skeletal muscle growth in individual live zebrafish subjected to altered electrical activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

A rapid and precise measure of somite volume 
A method of sample preparation, data acquisition, and volumetric analysis that allows the rapid measurement of muscle growth in zebrafish larvae is described. Muscle size can be measured in live animals using fish labeled on their plasma membranes with a membrane-targeted GFP (&#946;-actin:HRAS-EGFP) or&#160;mCherry (&#945;-actin:mCherry-CAAX). Larvae were transiently anesthetized using tricaine, mounted in low-melting-point agarose and...

Watch this Scientific Journal Video about Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity at JoVE.com

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

real-time-repeated-measurement-skeletal-muscle-growth-individual-live

Research

JoVE Journal

Developmental Biology

1.9K Views.  King’s College London. Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

Video: Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...