Molecular Neuroscience: A Laboratory Manual · 2014-08-18 · MOLECULAR NEUROSCIENCE ALABORATORYMANUAL EDITED BY Rusty Lansford Department of Radiology Children’s Hospital Los Angeles

MOLECULARNEUROSCIENCE

A LABORATORY MANUAL

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MOLECULARNEUROSCIENCE

A LABORATORY MANUAL

EDITED BY

Rusty LansfordDepartment of Radiology

Children’s Hospital Los AngelesKeck School of Medicine

University of Southern California

COLD SPRING HARBOR LABORATORY PRESSCold Spr ing Harbor , New York † www.csh lp ress .o rg




MOLECULAR NEUROSCIENCEA LABORATORY MANUAL

All rights reserved# 2014 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkPrinted in the United States of America

Publisher John InglisDirector of Editorial Development Jan ArgentineManaging Editor Maria SmitDevelopmental Editor Kaaren JanssenProject Manager Maryliz DickersonProduction Editor Joanne McFaddenProduction Manager Denise WeissDirector of Product Development & Marketing Wayne ManosCover Designer Mike Albano

Front coverartwork: Multiplexed high-resolution mapping of complex interlaced and overlapping mRNA expression patterns inthe developing mouse as imaged by confocal microscopy. A transgenic whole-mount embryo was fixed at E10.5 and subjectedto the hybridization chain reaction (HCR) protocol on p. 224. Three orthogonal DNA HCR amplifiers carrying spectrally dis-tinct fluorophores were used. Shown is a composite image of the cyan channel, HCR-Alexa488 targeting Tg(egfp); theyellow channel, HCR-Alexa546 targeting Twist1; and the magenta channel, HCR-Alexa647 targeting Pax2. Image by DavidHuss and Rusty Lansford.

Library of Congress Cataloging-in-Publication Data

Molecular neuroscience : a laboratory manual / edited by Rusty Lansford.

p. ; cm.

Includes bibliographical references and index.

ISBN 978-1-62182-013-0 (cloth) – ISBN 978-1-62182-014-7 (pbk.)

I. Lansford, Rusty.

[DNLM: 1. Genetic Phenomena–Laboratory Manuals. 2. GeneticTechniques–Laboratory Manuals. 3. Molecular Imaging–LaboratoryManuals. 4. Neurons–physiology–Laboratory Manuals. QU 25]

QP356.2

573.8’4076–dc232014023667

10 9 8 7 6 5 4 3 2 1

Students and researchers using the procedures in this manual do so at their own risk. Cold Spring Harbor Laboratory makes no repre-sentations or warranties with respect to the material set forth in this manual and has no liability in connection with the use of thesematerials. All registered trademarks, trade names, and brand names mentioned in this book are the property of the respective owners.Readers should please consult individual manufacturers and other resources for current and specific product information.

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Contents

Foreword xi

Preface xiii

SECTION 1. WORKING WITH CELLS, 1

Working without Contamination 3Kathy Barker

Sterile Pipetting and Pouring Techniques 6Kathy Barker

Filter Sterilization Techniques 9Kathy Barker

Aspirating Fluids with Sterile Technique 11Kathy Barker

Working Sterilely in a Biosafety Cabinet 13Kathy Barker

Mammalian Cell Culture 16Gretchen J. Darlington

Purification and Culture of Retinal Ganglion Cells 21Alissa Winzeler and Jack T. Wang

Purification and Culture of Retinal Ganglion Cells from Rodents 25Alissa Winzeler and Jack T. Wang

Purification and Culture of Astrocytes 35Lynette C. Foo

Purification of Rat and Mouse Astrocytes by Immunopanning 38Lynette C. Foo

Maintaining Live Cells and Tissue Slices in the Imaging Setup 50Michael E. Dailey, Glen S. Marrs, and Dana Kurpius

SECTION 2. WORKING WITH DNA, 59

BACTERIA BASICS

Bacteria 61Kathy Barker

Making Media for Bacterial Culture 66Kathy Barker

Obtaining Isolated Colonies of Bacteria 70Kathy Barker

Using a Petroff-Hausser Counting Chamber 73Kathy Barker

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Measurement of Bacterial Growth by Spectrophotometry 75Kathy Barker

Freezing Bacteria for Long-Term Storage 78Kathy Barker

DNA BASICS

Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate:Minipreparation 81Joseph Sambrook and David W. Russell

Preparation and Transformation of Competent E. coli Using Calcium Chloride 85Joseph Sambrook and David W. Russell

Quantitation of DNA and RNA 89Carlos F. Barbas III, Dennis R. Burton, Jamie K. Scott, and Gregg J. Silverman

The Basic Polymerase Chain Reaction 91Joseph Sambrook and David W. Russell

Optimization and Troubleshooting in Polymerase Chain Reaction 94Kenneth H. Roux

Working with Bacterial Artificial Chromosomes 100Joseph Sambrook and David W. Russell

Isolation of Bacterial Artificial Chromosome DNA from Small-Scale Cultures 103Joseph Sambrook and David W. Russell

Homologous Recombination Using Bacterial Artificial Chromosomes 106Cary Lai, Tobias Fischer, and Elizabeth Munroe

Bacterial Artificial Chromosome Transgenic Mice and the GENSAT Database ofEngineered Mouse Strains 117Eric F. Schmidt, Laura Kus, Shiaoching Gong, and Nathaniel Heintz

Agarose Gel Electrophoresis 124Joseph Sambrook and David W. Russell

Southern Blotting: Capillary Transfer of DNA to Membranes 128Joseph Sambrook and David W. Russell

Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilizedon Membranes 134Joseph Sambrook and David W. Russell

PROTEIN–DNA INTERACTIONS

Electrophoretic Mobility-Shift Assays 139Michael F. Carey, Craig L. Peterson, and Stephen T. Smale

Chromatin Immunoprecipitation (ChIP) Analysis of Protein–DNA Interactions 143Tae Hoon Kim and Job Dekker

Formaldehyde Cross-Linking 147Tae Hoon Kim and Job Dekker

Preparation of Cross-Linked Chromatin for ChIP 150Tae Hoon Kim and Job Dekker

ChIP 153Tae Hoon Kim and Job Dekker

ChIP-Quantitative Polymerase Chain Reaction (ChIP-qPCR) 158Tae Hoon Kim and Job Dekker

Contents

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ChIP-chip 160Tae Hoon Kim and Job Dekker

ChIP-seq 167Tae Hoon Kim and Job Dekker

SECTION 3. WORKING WITH RNA, 173

RNA BASICS

The Fundamentals of RNA Purification 175Timothy W. Nilsen

General Procedures for Avoiding Contamination with RNase 182Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer

Purification of RNA from Cells and Tissues by Acid Phenol-GuanidiniumThiocyanate-Chloroform Extraction 183Joseph Sambrook and David W. Russell

Isolating Total RNA from Mouse Embryos or Fetal Tissues 187Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer

Single-Neuron Isolation for RNA Analysis Using Pipette Capture and LaserCapture Microdissection 191Ditte Lovatt, Thomas Bell, and James Eberwine

Antisense RNA Amplification for Target Assessment of Total mRNA froma Single Cell 200Jacqueline Morris, Thomas J. Bell, Peter T. Buckley, and James H. Eberwine

Denaturation and Electrophoresis of RNA with Formaldehyde 212Donald C. Rio

Northern Blots: Capillary Transfer of RNA from Agarose Gels and FilterHybridization Using Standard Stringency Conditions 216Donald C. Rio

Combinatorial Analysis of mRNA Expression Patterns in Mouse Embryos UsingHybridization Chain Reaction 224David Huss, Harry M.T. Choi, Carol Readhead, Scott E. Fraser, Niles A. Pierce,and Rusty Lansford

Electrophoretic Mobility Shift Assays for RNA–Protein Complexes 234Donald C. Rio

RNase Footprinting to Map Sites of RNA–Protein Interactions 240Timothy W. Nilsen

Identification of RNA Cargoes by Antibody-Positioned RNA Amplification 246Kevin Y. Miyashiro and James Eberwine

CLIP (Cross-Linking and Immunoprecipitation) Identification of RNAs Boundby a Specific Protein 254Robert Darnell

TRANSCRIPTOME ANALYSIS

Transcriptome Analysis Using Microarrays

Preparation of Fluorescent-Dye-Labeled cDNA from RNA for MicroarrayHybridization 269Manuel Ares Jr.

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Microarray Slide Hybridization Using Fluorescently Labeled cDNA 274Manuel Ares Jr.

Scanning Microarray Slides 280Manuel Ares Jr.

Tips on Hybridizing, Washing, and Scanning Affymetrix Microarrays 285Manuel Ares Jr.

Methods for Processing Microarray Data 291Manuel Ares Jr.

Transcriptome Analysis with High-Throughput Sequencing

Fragmentation of Whole-Transcriptome RNA Using E. coli RNase III 296Manuel Ares Jr.

Preparation of Small RNA Libraries for High-Throughput Sequencing 299Colin Malone, Julius Brennecke, Ben Czech, Alexei Aravin, and Gregory J. Hannon

Tips for Preparing mRNA-Seq Libraries from Poly(A)þ mRNA for IlluminaTranscriptome High-Throughput Sequencing 310Brenton R. Graveley

High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation 313Silin Zhong, Je-Gun Joung, Yi Zheng, Yun-ru Chen, Bao Liu, Ying Shao, Jenny Z.Xiang, Zhangjun Fei, and James J. Giovannoni

Methods for Processing High-Throughput RNA Sequencing Data 323Manuel Ares Jr.

RNAi

Creating an miR30-Based shRNA Vector 333Kenneth Chang, Krista Marran, Amy Valentine, and Gregory J. Hannon

Packaging shRNA Retroviruses 338Kenneth Chang, Krista Marran, Amy Valentine, and Gregory J. Hannon

Infection of Mammalian Cells with Retroviral shRNAs 342Kenneth Chang, Krista Marran, Amy Valentine, and Gregory J. Hannon

Creating Transgenic shRNA Mice by Recombinase-Mediated Cassette Exchange 344Prem K. Premsrirut, Lukas E. Dow, Youngkyu Park, Gregory J. Hannon,and Scott W. Lowe

SECTION 4. GENE TRANSFER, 353

Nonviral Methods

DNA Transfection Mediated by Lipofection 355Joseph Sambrook and David W. Russell

Transfection of Mammalian Cells with Fluorescent Protein Fusions 358David L. Spector and Robert D. Goldman

DNA Transfection by Electroporation 364Joseph Sambrook and David W. Russell

Contents

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PiggyBac Transposon-Mediated Cellular Transgenesis in Mammalian Forebrainby In Utero Electroporation 367Fuyi Chen, Brady J. Maher, and Joseph J. LoTurco

Single Cell/Cellular Subregion-Targeted Phototransfection 376Jaehee Lee, Jai-Yoon Sul, James H. Eberwine

VIRAL METHODS

Generation and Analysis of Lentivirus Expressing a 2A Peptide-LinkedBicistronic Fluorescent Construct 381David Huss and Rusty Lansford

Lentiviral Vectors for Retrograde Delivery of Recombinases and Transactivators 403Ian R. Wickersham, Heather A. Sullivan, Gerald M. Pao, Hiroki Hamanaka,Ki A. Goosens, Inder M. Verma, and H. Sebastian Seung

Rabies Viral Vectors for Monosynaptic Tracing and Targeted TransgeneExpression in Neurons 410Ian R. Wickersham and Heather A. Sullivan

Concentration and Purification of Rabies Viral and Lentiviral Vectors 421Heather A. Sullivan and Ian R. Wickersham

Stable Producer Cell Lines for Adeno-Associated Virus (AAV) Assembly 427Gilliane Chadeuf and Anna Salvetti

Generation of Replication-Competent and -Defective Herpes simplexVirus (HSV) Vectors 432William F. Goins, David M. Krisky, James B. Wechuck, Darren Wolfe, Shaohua Huang,and Joseph C. Glorioso

Construction and Packaging of Herpes simplex Virus/Adeno-AssociatedVirus (HSV/AAV) Hybrid Amplicon Vectors 441Okay Saydam, Daniel L. Glauser, and Cornel Fraefel

SECTION 5. IMAGING, 447

MICROSCOPY BASICS

Microscopy 449Kathy Barker

Using the Light Microscope 453Kathy Barker

Confocal Microscopy: Principles and Practice 463Alan Fine

Principles of Multiphoton-Excitation Fluorescence Microscopy 471Winfried Denk

Digital Scanned Laser Light Sheet Fluorescence Microscopy 476Philipp J. Keller and Ernst H. K. Stelzer

LIGHT MODULATION OF PROTEINS

Constructing and Expressing Fluorescent Protein Fusions 489David L. Spector and Robert D. Goldman

Contents

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Imaging Green Fluorescent Protein-Labeled Neurons Using Light and ElectronMicroscopy 492Graham W. Knott

Imaging Synaptic Protein Dynamics Using Photoactivatable GreenFluorescent Protein 501Robby M. Weimer, Travis C. Hill, Andrew M. Hamilton, and Karen Zito

Imaging Neuronal Activity with Genetically Encoded Calcium Indicators 508Lin Tian, S. Andrew Hires, and Loren L. Looger

Measuring Membrane Voltage with Fluorescent Proteins 518Jordan Patti and Ehud Y. Isacoff

Optogenetics: Opsins and Optical Interfaces in Neuroscience 526Antoine R. Adamantidis, Feng Zhang, Luis de Lecea, and Karl Deisseroth

Establishing a Fiber-Optic-Based Optical Neural Interface 534Antoine R. Adamantidis, Feng Zhang, Luis de Lecea, and Karl Deisseroth

IN VIVO IMAGING

Single-Cell Electroporation in Xenopus 540Xue Feng Liu and Kurt Haas

Single-Cell Electroporation of Xenopus Tadpole Tectal Neurons 546Xue Feng Liu and Kurt Haas

In Vivo Time-Lapse Imaging of Neuronal Development in Xenopus 552Edward S. Ruthazer, Anne Schohl, Neil Schwartz, Aydin Tavakoli, Marc Tremblay,and Hollis T. Cline

Bulk Electroporation of Retinal Ganglion Cells in Live Xenopus Tadpoles 558Edward S. Ruthazer, Anne Schohl, Neil Schwartz, Aydin Tavakoli, Marc Tremblay,and Hollis T. Cline

4D Fluorescent Imaging of Embryonic Quail Development 563Christie A. Canaria and Rusty Lansford

Preparation and 4D Fluorescent Imaging of Quail Embryos 567Christie A. Canaria and Rusty Lansford

Generating and Imaging Multicolor Brainbow Mice 575Tamily A. Weissman, Joshua R. Sanes, Jeff W. Lichtman, and Jean Livet

Two-Photon Imaging of Microglia in the Mouse Cortex In Vivo 583Axel Nimmerjahn

Mapping Anatomy to Behavior in Thy1:18 ChR2-YFP Transgenic MiceUsing Optogenetics 594Lief E. Fenno, Lisa A. Gunaydin, Karl Deisseroth

APPENDIX

General Safety and Hazardous Material Information 607

INDEX 613

Contents

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Foreword

I remember sitting in my office at Stanford in the fall of 1985 and receiving the call from Susan Hock-feld asking me to start a summer course at Cold Spring Harbor Laboratory that utilized molecularbiology techniques. Sue was a developmental neuroscientist who was on staff at CSHL at that timeand she was in charge of the summer courses as well. In talking with Sue about the remit of the course,she indicated that the techniques of molecular biology were slowly coming into the neurosciences andshe thought that a laboratory course would help speed this up. I had never attended a course at CSHL;and while Sue said that they were easy to do because of the quality of students, I really had no idea whatI was in for (it is my guess that none of the directors that followed me knew exactly what they were infor, either). My colleagues, Drs. Beth Schachter (then at Mt. Sinai) and Marian Evinger (at StonyBrook), agreed to co-direct the course that first year. At that point the emphasis was on cloning ofneural genes (hence the original name of the course, “Molecular Cloning of Neural Genes”) andthe students isolated RNA, made cDNA libraries, screened the libraries, isolated clones and sequencedthem. Other methodologies that were taught were in situ hybridization, DNA isolate, restriction map-ping, etc.

That first year I remember walking along Bungtown Road with Marian around three in the after-noon when none of the cDNA libraries turned out, bemoaning our failure and trying to figure outhow to move forward. As anyone who has attended this course or any of the other courses knows, itis usually the failures that bring out the most discussion and the greatest opportunity to learn thenitty-gritty of the techniques that are being taught.

Over the nearly 30 years of its existence, the course directors and instructors have changed butthe remit of teaching the latest molecular techniques to neuroscientists is still important and hasbeen expanded to include more cell biological experiments. This is reflected in the transitionsfrom the directors who were molecular biologists, including myself, Beth Schacter, Marian Evinger,Moses Chao, Robert Margolskee, Robert Darnell, and of course Jim Boulter. Boulter was director fortwo different stretches of time and was an exemplary “librarian.” He brought Cary Lai into the foldas director, and Cary has the remarkable record for the most continuous summers of co-directingthe course at 23. It was during Boulter’s directorship that the course name changed to “AdvancedTechniques in Molecular Neuroscience,” reflecting the advances of genomics and the fact that mostgenes had been cloned, sequenced, and were available for use.

The course curriculum changed as molecular neuroscience evolved, as illustrated during thedirectorship of Thomas Hughes (Montana State) who introduced manipulation of green fluorescentprotein as a major emphasis of the course. Of course, microscopy has been the bulwark of neuro-science since the time of Cajal, and its marriage with molecular biology was ably conveyed to thecourse students during the directorship of Rusty Lansford (editor of this manual), who made livecell imaging and ways of manipulating live cells a major emphasis of the course.

Consistent with the director’s vision for the course, there were many instructors who broughttheir technologies to the course and were generous in their time, giving didactic lectures and pro-viding hands-on teaching of their sections, which typically ran from a couple of days to a week ormore. There were also many lecturers who came to give an evening research seminar in the course.The lecturers came from many institutions, ranging in experience from advanced post-docs to NobelLaureates. Some of the lecturers truly reveled in the opportunity to lecture in the course, as any stu-dent will tell you of Robert Darnell’s lectures, which always exceeded 3 h.

For the directors, instructors, and lecturers, the highlight of course attendance was interactingwith the students. The students came from many countries and with differing levels of experience;some were grad students and others were members of the U.S. National Academies of Science. They

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were united in their desire to learn the newest technologies and to be able to apply these method-ologies to their own scientific questions. While it is clear that the technical details were important,the students also learned how to think about the techniques they were employing—the sensitivity ofthe methodologies, whether the question can be answered using these technologies, and how thedata might be interpreted. These insights separate this course from many of the other courses taughtaround the world. It is this “ATMN/CNGEN effect” that has enabled the course participants to uti-lize the course in ways that significantly benefit their work, and through this, scientific advancement.

The course is still running strong each summer, teaching 16 lucky students selected to partici-pate. Beyond these students, however, are many others who would benefit from detailed knowledgeof the most current techniques. This manual is intended to provide this opportunity to the manywho can’t attend the course. Rusty has done an exceptional job of bringing together past directorsand instructors as well as former students to provide detailed protocols for the cutting-edge sciencethey do that reflect and expand upon the continuing goals of the course.

James H. Eberwine, PhDUniversity of Pennsylvania School of Medicine,

Philadelphia, PA

Foreword

xii




Preface

The molecular genetics revolution has significantly impacted all fields of biological and medicalresearch. The ability to manipulate and analyze DNA, RNA, and proteins permits investigationsof cells, tissues, and organisms using approaches unimagined a generation ago. We are currentlyin the midst of another technical revolution, in which cells may be dynamically viewed and manip-ulated with laser-based tools; organismal genomes, transcriptomes, proteomes, and other ‘omes’ canbe acquired overnight; and novel methods are being developed by interdisciplinary teams to quan-titate the massive amounts of data emerging from such experiments. The modern neuroscientist istasked with understanding how these interdisciplinary tools and techniques may be synergisticallyintegrated to investigate the inner workings of normal and diseased nervous systems.

I have had the pleasure to teach and direct the Advanced Techniques in Molecular Neuroscience(ATMN) course at Cold Spring Harbor Laboratory (CSHL) for the past decade. Recently my col-leagues and I decided that it would be helpful to pull together the protocols that have been taughtand tested at ATMN over this period to produce a laboratory manual that would have broad use forneuroscientists beyond this course. This manual, Molecular Neuroscience, primarily draws from theprotocols used in the various modules of the ATMN course, as well as those of its predecessor,Molecular Cloning of Neural Genes. By necessity and design, the ATMN course is constantly evolv-ing to keep pace and teach the most advanced techniques in molecular neuroscience in order to pre-pare the next generation of neuroscience researchers to fearlessly and imaginatively develop the next-next generation of techniques and technologies. As the field of neuroscience changes, driven byemerging tools and technologies, so do the instructors and the protocols, as reflected here in Molec-ular Neuroscience. ATMN course instructors and lecturers, internationally recognized as experts invarious fields of molecular neuroscience, wrote many of the protocols in this manual.

The ATMN course comprises three components: an extensive and up-to-date set of laboratoryexercises, lectures on theoretical and practical aspects of advanced molecular biological and imagingtechniques, and lectures by invited speakers that serve to showcase the ways in which these molec-ular techniques have been used to advance specific areas of neuroscience. The laboratory portion ofeach course includes the following topics: in vitro and in vivo gene delivery systems; loss-of-functionapproaches (e.g., RNAi) to study gene function; optogenetics to probe neural circuits within ani-mals; the use of RNA-seq and microarray analyses to profile gene expression; methods to quantitategene expression in single cells; and techniques to isolate, propagate, and analyze desired neuronaland glial cell populations. As many of these advanced techniques also involve the use of more stand-ard procedures to first modify and then ‘move’ DNA fragments from one vector to another, the lab-oratory exercises also provide training in these methods, which are essential for the successfulexecution of the more sophisticated approaches. Advanced epifluorescence and confocal imagingmicroscopy and image analysis are often used to assay the various experiments, thereby educatingthe students in additional emerging technologies. Finally, course participants are introduced to bio-informatics analysis of microarray or high-throughput sequencing data sets.

These topics are all captured within the protocols and discussions that make up the MolecularNeuroscience lab manual. The book is divided into five sections. Section 1 begins with an introduc-tion to tissue culture using sterile techniques and soon moves on to culturing primary neural cells.Tissue culture is an essential technique in neuroscience and is fundamental for many of the proto-cols found in the rest of the manual—for collecting and assaying adequate and intact samples ofDNA (Section 2) and RNA (Section 3), for cell transfection and infection to deliver genetic payloadsinto cells and tissues (Section 4), and for imaging living cells and tissues (Section 5). Section 2 ini-tiates with the basics of growing bacteria and manipulating DNA before advancing into transgenic

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and ChIP techniques. Section 3 has numerous protocols to assay RNA complexity using standardand emerging approaches including microarray analysis and RNA-seq. The fluctuating transcrip-tome can be reliably assayed in single cells and assembled tissues, which is transforming the ques-tions pursued by basic researchers along with how clinicians diagnose and treat diseases. Section 4introduces and guides the reader through various gene transfer techniques in vitro and in vivo. Sec-tion 5 describes several types of microscopes increasingly being used to observe and perturb cells atunprecedented spatiotemporal resolution, drawing on Sections 1–4 to pull it off.

Each section begins with a brief introduction into the issues to be considered. The sections con-tinue with protocols for the basic techniques that must be mastered before moving on to the moreadvanced techniques at the end of the sections. The advanced protocols draw heavily from the bleed-ing edge of science and often require multidisciplinary approaches. The protocols concisely explainrequired reagents and equipment before delving into the experimental steps. They include discus-sions and notes on troubleshooting potential problems that may arise during the experiment orwith the interpretation of the data. As with any new protocol or recipe, users are strongly advisedto familiarize themselves with the procedure prior to beginning.

Molecular Neuroscience is intended to provide neuroscientists at all levels with a conceptual andpractical understanding of key techniques in molecular neuroscience. Based on its advanced content,scholars that range from advanced undergraduate students to laboratory heads will find it indispen-sable. A number of the protocols and discussions were drawn from other CSHL Press manuals andthe online methods journal Cold Spring Harbor Protocols.

This manual owes an enormous debt of gratitude to the ATMN students who spent weeks ofsleepless nights attempting the protocols. They helped the instructors to fine-tune and troubleshootthe methods, contributing to the evolution of this manual’s protocols. Most of them will acknowl-edge that their end-of-course exhaustion was due to time very well spent—even if they did notappreciate it for 6 or so months. I think one of the most valuable aspects of the course, one thatI hope will be used by the readers of this book, is the willingness to overcome the activation barriersand fear and try new protocols, to boldly go where few have gone, and to follow and develop novelprotocols that bypass experimental roadblocks. ATMN is a place where the instructors and lecturersexpect the students to talk science with them into the wee hours. It is a place where the students areexpected to embrace and exercise their inner geek.

I am indebted to my predecessors Jim Eberwine, Moses Chao, Robert Margolskee, Jim Boulter,Bob Darnell, Thom Hughes, and currently Joe Loturco for establishing a course with high expect-ations and standards. Jim Eberwine’s creativity and support are unparalleled and greatly appreciated.Worth special note is Cary Lai, who has been involved with the course for over 20 years as a student,teaching assistant, and instructor, for continually teaching me his patient and detailed teaching style.David Huss—my right and left hand in the lab—helped in innumerable ways. Before I even ask, Ialmost always find that he has done what is needed, calmly making difficult experiments work seam-lessly, helping all who need help. The editorial staff at CSHL Press have been great to work with andextremely supportive and guided me with a gentle hand. Of special note are Maria Smit, JoanneMcFadden, and Maryliz Dickerson. Finally, I thank my son Jack who has always very lovingly, albeitreluctantly, supported my need to leave for CSHL each summer to teach at ATMN. I always returntired and spent, fully in need of his perceptive insistence to come out and play.

Rusty LansfordMay 2014

Preface

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General Safety and Hazardous Material Information

This manual should be used by laboratory personnel with experience in laboratory and chemicalsafety or students under the supervision of such trained personnel. The procedures, chemicals,and equipment referenced in this manual are hazardous and can cause serious injury unless per-formed, handled, and used with care and in a manner consistent with safe laboratory practices.Students and researchers using the procedures in this manual do so at their own risk. It is essentialfor your safety that you consult the appropriate Material Safety Data Sheets, the manufacturers’manuals accompanying equipment, and your institution’s Environmental Health and SafetyOffice, as well as the General Safety and Disposal Cautions in the Appendix, for proper handlingof hazardous materials in this manual. Cold Spring Harbor Laboratory makes no representations orwarranties with respect to the material set forth in this manual and has no liability in connectionwith the use of these materials.

All registered trademarks, trade names, and brand names mentioned in this book are the prop-erty of the respective owners. Readers should please consult individual manufacturers and otherresources for current and specific product information. Appropriate sources for obtaining safetyinformation and general guidelines for laboratory safety are provided in the General Safety andHazardous Material Information Appendix at the end of this manual.







Documents

Molecular Neuroscience: A Laboratory Manual · 2014-08-18 · MOLECULAR NEUROSCIENCE ALABORATORYMANUAL EDITED BY Rusty Lansford Department of Radiology Children’s Hospital Los Angeles