We thank participating children and families, all staff at the Children's Hospital Boston (CHB) with special appreciation to the MRI technicians, without whom our studies could not be conducted. We are especially grateful to Arnold Cyr for participation and help during our video shoot and to operation manager Patricia Devine at the CHB Waltham. Thanks to the research and technical staff at MIT's Athinoula A. Martinos Imaging Center for sharing experience in MR image acquisition and analysis.
This research was funded by the Charles H. Hood Foundation, a Children's Hospital Boston pilot grant and the Swiss National Foundation (N.M.R.)

We have incorporated general experimental testing guidelines as well as MRI specific approaches 12-23 into one complete neuroimaging protocol intended to guide researchers and clinicians during neuroimaging sessions with awake children as young as four years of age. First, we aim to emphasize general testing guidelines adapted for MRI examinations. Second, we provide a hands-on, step-by-step description of our neuroimaging protocol. In our experience, a single session of approximately 2.5 hours (including a maximal imaging time of 45-60 minutes) is sufficient to train and guide a child through a neuroimaging session.
General Guidelines
As in every testing session with pediatric populations, general guidelines and recommendations for how best to work with young children should be considered 24. We highlight comfort, appropriateness and motivation (CAM) and provide definitions of these concepts.
(C) Comfort: Comfort is defined as the emotional state of a young participant involved in an imaging session where the feeling of threat is minimized and security is maximized.
Environment: In line with other research groups 19,20, we consider the mock scanner area an ideal place to start a neuroimaging session. Ideally, a mock scanner area replicates the actual MRI room and MR scanner to the greatest extent possible (e.g. including a mock MR scanner mirroring an actual MR scanner's appearance and the sounds produced) 20. This room provides the same equipment (e.g. response tools) as the actual MRI room. Without a static magnetic field, it is a safe place to familiarize the child with the imaging procedure in a child-appropriate way. The mock scanner area can be designed in a child-friendly manner by adding stuffed animals, placing a few toys in the room (not too many because this could be distracting), having parents and siblings sit on child-size chairs and table, and providing some parent-approved snacks and drinks. We further provide the child with a sticker chart, a CD with the child's structural brain images, and a treasure chest with a gift certificate and other small prizes.
Family &amp; Friends: One way to facilitate active participation during a neuroimaging session is to encourage the participating children to invite their family, siblings and friends or to bring their own stuffed animals or toys. In addition, children and their parents should be allowed to choose whether they prefer to have one parent be present in the MRI room during the fMRI experiment.
Clothing: Clothing with no metallic pieces (e.g., buttons or zippers) is mandatory. Children may prefer to wear their own clothing. However, appropriate attire (e.g. hospital gowns) should be available if needed. As training and MRI rooms can be cold, a blanket may offer the child additional comfort.
(A) Appropriateness: Appropriateness describes the frameworks and contexts used to present activities and materials during the neuroimaging session in relation to the age group studied. 
Terminology: The terminology and practices used during pediatric neuroimaging sessions should be carefully chosen. Doing so can avoid misconceptions or a frightening atmosphere. Inappropriate terms, for example, include the following: "it is really loud, but it will not hurt you" or "how are you doing inside the machine?. It is recommended to use positive language that is easily understood by children and to address potential issues directly. Avoiding certain phrases can be advantageous for the session and a child-appropriate choice of technical terms is recommended (e.g. "brain camera" instead of "MRI machine", "camera click" instead of "scanner noise", etc.).
Misconceptions: One important goal is to clarify the child's misconceptions about imaging as early as possible during the neuroimaging session. An easily understandable study description should be communicated prior to the visit and may be repeated at the beginning of the neuroimaging session.
Response Tools: Using specific response tools appropriate to the age of the participant has proven to be beneficial 25. As an example, when using various instruments (e.g. headphones, response buttons, eye tracker, etc.), it is important to use those that are child-appropriate in size and shape. These instruments should be carefully positioned so that the child does not wiggle and move in an attempt to reach the response devices.
Affective State: As in every testing session, it is essential to be sensitive to the affective state of the participant. Children may not always express their feelings readily, but anxiety, boredom or frustrations need to be recognized and dealt with promptly 7,24. Concerns need to be addressed directly and questions need to be posed in a child-appropriate manner.
Flexible Approach: It is highly recommended to account for the specific needs of each child and to allow sufficient time to make a flexible approach possible (e.g. optional help depending on the child, additional training sessions, allowing enough time for the neuroimaging session, etc.).
(M) Motivation: Motivation describes the willingness of a participant to actively cooperate within a given research setting.
Child-Friendly Themes: We recommend using a fun theme that guides children throughout the neuroimaging session (e.g. an adventure story). By doing so, the children become invested and engaged in the sequence of the activities. Furthermore, a theme can give the research team an opportunity to build all experiments and task paradigms in a child-friendly way (e.g. using cartoon characters).
Traditional &amp; Virtual Sticker Charts: Sticker charts are well known as tools to help motivate children to complete different experiments within one session. A virtual sticker chart is analogous to the traditional sticker chart, but it can be shown (via projector) to the child when inside the MR scanner. As an example, we use a virtual sticker chart modeled after the children's game "Chutes and Ladders", where the participants have to find their way home (this can only be achieved by completing the experimental tasks). Not only is this fun for the child, but it gives the research team time after each run (image acquisition time for one experimental task) to prepare for the next.
Course of Action
Pediatric Neuroimaging Session I: Preparation
1) Before the neuroimaging session: In preparation for the neuroimaging session, the participating families are provided with information through print or online media, which contain age-appropriate descriptions offering a preview of the study content and details. 
Pediatric Neuroimaging Session II: Training (approximately 1 hour)
2) Welcome &amp; paperwork: The pediatric neuroimaging session starts in the mock scanner area, where the research team welcomes the participating child, family and friends. Institutional review board (IRB) forms (e.g., consent and assent forms), MR scanner screening forms, and reimbursement procedures are reviewed and it is ensured that the scope of the neuroimaging session is clear for the child and the parents.
3) (f)MRI introduction: The mock scanner area is introduced to the child and the parents including a short description of the mock scanner, the response tools and additional material used during the neuroimaging session. A digital camera can be used to explain how a regular camera takes pictures and how a "brain camera" (MRI scanner) works. Examples of sharp and blurry pictures demonstrate the impact of motion on the quality of the photos or pictures (see Figure 1). Movement restriction may be illustrated through the use of optional games, such as the "Freezing-Game" or "Statue-Game". These games require the child to stay very still (e.g. as an ice sculpture or statue) for a short period of time.
<img src="/files/ftp_upload/1309/1309fig1.jpg" alt="figure 1" /> Figure 1. (A) Sharp and (B) blurry pictures may be used to demonstrate the impact of motion on picture quality.
4) Introduction to experiment: Before introducing the experimental tasks one can start with a short enjoyable activity. Next, the use of the mock scanner is slowly incorporated.
5.1) Short movie: For this experiment a child friendly theme is added to the neuroimaging session. The children take part in a spaceship adventure game which is introduced by a short movie; the main characters guide the child through all experiments.
5.2) Experimental tasks / "games": The use of the mock scanner is incorporated step-wise: (1) instructions are shown while the child is sitting on the mock scanner bed; (2) the instructions are repeated using printed cards which mirror a computer screen; (3) the first few examples are solved with the help of the research team; (4) training items are then presented in real time; (5) as a next step the child is offered the use of headphones and pre-recorded scanner background sounds are played back during the presentation of the training items; (6) finally, the child is allowed to play the game inside the mock scanner. The research team ensures understanding of the experimental tasks.
If using more than one response tool, such as one button in each hand, it is important to define which response tool corresponds to which answer. A child may not know the difference between left and right. A stuffed animal (e.g. a monkey) can be placed next to one side of the child. Instead of the instructions "press the right button", the instructions are changed to "press the monkey button".
5) Rules of communication: During the training the child is taught that a gentle hand press on their leg signals that they are moving too much. This signal serves as a means of communication possibility between the research team and the child during the actual fMRI experiment. It is explained to the child that speech should be avoided during image acquisition as it could interfere with the experimental task performance and impact data quality.
6) Short break / dice game: During a break, dice may be used to determine a random order in which the games will be played during the fMRI experiment. This ensures randomization across participants.
7) Repetition of main points: Before starting the fMRI experiment it is recommended that the researcher repeat the most important rules for the neuroimaging session to the child. In order to minimize memory demands on young children, it is helpful to summarize critical information in a few main points and to reiterate those throughout the session.
8) Trouble shooting: When a child hesitates in taking part in the training session or fMRI experiment, researchers are advised to (1) act immediately; (2) offer to take a break; (3) address concerns and assure that participation is voluntarily; and depending on the child's choice the session is either stopped early or (4) game strategies as recommended by play therapists 12,26 are used to help a more anxious child get comfortable with the new environment. It is important to use flexible approaches that allow a child to participate at a pace that is comfortable for them. However, the child's choice in participation needs to be evident.
9) MRI training without mock scanner: If researchers or clinicians do not have access to a mock scanner area, game strategies as used by play therapists are highly recommended 26. For example, a neuroimaging session can be simulated by using pictures or a model of an MR scanner 12. Furthermore, behavioral approaches using desensitization and operant behavioral techniques 13-15,17,23 have helped to reduce distress in children and increase the number of children successfully completing their neuroimaging session.
Pediatric Neuroimaging Session III: Break and Metal Screening
10) Metal screening: Before entering the MRI room, every participant and parent must fill out and sign an MRI screening form. Additionally, the child needs to be checked for ferrous objects. A hand held metal detector or a magnet can be used to introduce the screening playfully (e.g. "pretend we are at the airport").
Pediatric Neuroimaging Session IV: fMRI Experiment (45 to 60 min)
General: During the fMRI experiment, the biggest challenge for many children is to stay very still for a long period of time. It is recommended to keep each experiment as short as possible or to divide the experiment into two experimental runs. In our experience, run imaging times with a maximum of five to seven minutes have proven accomplishable by children elementary school age or younger. The total length of the imaging session should also be considered. However, it is easier for a child to go through a long imaging session with several short experimental runs than to complete a shorter session with longer experimental runs. Neuroimaging experiments often employ a block design in which different experimental task conditions are presented in a series of subsequent blocks. Children can easily get confused by changing task demands within one experimental run and therefore it is recommended to keeping task conditions separated (e.g., by sampling two different task conditions in two subsequent experimental runs with a break in between).
11) Facilitate transition: To facilitate the child's transition to the actual MRI room, the child is accompanied by a research staff member and may choose to bring along a parent and the child's favorite stuffed animal, as long as the toy does not contain any ferrous parts. Additionally one research team member is recommended to accompany the child.
12) Equipment check: Screen and mirror positioning (for visual experiments), sound volume (for auditory experiments), and response tools need to be checked and ear protection (e.g., ear plugs) should be provided for attendants in the MRI room. Check the response tools and settings with applied examples (e.g. play questions for the child to test audio settings and have the child answer them). The research team must ensure that every family member present in the MRI room received information on appropriate behavior and rules, especially concerning safety.
13) fMRI / experimental task performance: Task instructions should be repeated before the start of each game (experimental task). After each game, the virtual sticker chart rewards the child's cooperation. While in the MR scanner, the child's comfort and movement must be monitored. The presence of a research team member in the MRI room has proven to be highly advantageous; the child's well-being and behavior can be observed, and essential communication and motivating feedback can be given directly to the child. Head movement of the child may be observed by the research team member present in the MRI room or by the researcher monitoring the image acquisition outside of the MRI room. A gentle hand press practiced during the training session signals to the child that they are moving too much. For experiments with several experimental runs, a short break half way through the neuroimaging session maintains comfort. As a motivational factor during the break, the child can be shown his or her own brain images.
14) Structural image acquisition: Finally structural MR image acquisition requires the participant to lie still without performing any experimental task. The child can be entertained by watching a short movie during this time. Recommended examples of appropriate movies are animal movies or documentaries as they are enjoyable and unlikely to induce laughing which may lead to head movements during image acquisition. The child handles movement restriction during the time of image acquisition more easily if told that pictures acquired during this time will be presented to them as a gift to take home, such as a CD with the child's brain pictures.
15) Closing: Once image acquisition is complete the reimbursement procedure follows including gifts, prizes and a CD with acquired brain pictures for the child to take home.
Representative Results
The use of appropriate preparation protocols and child-friendly imaging procedures positively influences cooperation, motivation and the experience of our participating children and their families. Use of this protocol reduces overall movement and thereby increases the chance of obtaining high quality images without the use of sedation or GA.
Using the current protocol we have recently obtained functional and structural brain imaging data for a group of pre-school aged children (ages 4.9 to 6.3 years / average age 5.5 years). Over 95% of all children have been able to complete a neuroimaging session including mock scanner training and fMRI experiment. The guidelines and procedures presented in this protocol were designed for pediatric neuroimaging sessions. However the general principle and many of the described tools can be applied to pediatric imaging sessions in general such as image acquisition of other body parts.

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C., Pike, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Event-related+fMRI+of+the+auditory+cortex.">Event-related fMRI of the auditory cortex.</a> Neuroimage. 10 (4), 417-429 (1999).</li><li>Gaab, N., Gaser, C., Zaehle, T., Jancke, L., Schlaug, G., G, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+of+pitch+memory--an+fMRI+study+with+sparse+temporal+sampling.">Functional anatomy of pitch memory--an fMRI study with sparse temporal sampling.</a> Neuroimage. 19 (4), 1417-1426 (2003).</li><li>Hall, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Sparse"+temporal+sampling+in+auditory+fMRI.">"Sparse" temporal sampling in auditory fMRI.</a> Hum Brain Mapp. 7 (3), 213-223 (1999).</li><li>Gaab, N., Gabrieli, J. D., Glover, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+in+peace+or+noise:+scanner+background+noise+suppresses+default-mode+network.">Resting in peace or noise: scanner background noise suppresses default-mode network.</a> Hum Brain Mapp. 29 (7), 858-867 (2008).</li></ol>

The authors have nothing to disclose.

The emergence of functional and structural MRI to study the human brain has facilitated possibilities of examining typical as well as atypical brain structure and functions and therefore holds great promise for both research and clinical purposes 6. However, MRI studies in younger age groups remain less numerous when compared to those of adults, adolescents or infants, which is mainly due to technical and practical difficulties when performing pediatric neuroimaging sessions. The curr...

making mr imaging child's play - pediatric neuroimaging protocol, guidelines and procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12, behavioral approaches 13, 14,15, 16-18 and simulation 19, the use of mock scanner areas 20,21, basic relaxation 22 and a combination of these techniques 23 have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.

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Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

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.

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

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18.1K Views.  Children’s Hospital Boston. 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. 

Video: Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...