Drosophila protocols

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Cold Spring Harbor Laboratory Press, 2000 - Medical - 697 pages
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This exceptional laboratory manual describes thirty-seven procedures most likely to be used in the next decade for molecular, biochemical, and cellular studies on Drosophila. They were selected after extensive consultation with the research community and rigorously edited for clarity, uniformity, and conciseness.The methods included permit investigation of chromosomes, cell biology, molecular biology, genomes, biochemistry, and development. Each protocol includes the basic information needed by novices, with sufficient detail to be valuable to experienced investigators. Each method is carefully introduced and illustrated with figures, tables, illustrations, and examples of the data obtainable. The book's appendices include key aspects of Drosophila biology, essential solutions, buffers, and recipes.An evolution of Michael Ashburner's 1989 classic Drosophila: A Laboratory Manual, this book is an essential addition to the personal library of Drosophila investigators and an incomparable resource for other research groups with goals likely to require fly-based technical approaches.

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Contents

In Situ Hybridization to Somatic Chromosomes
25
Restriction Enyzme Fragmentation ot Probe DNA
32
Secondary DOPPCR Amplification
39
Formaldehyde Fixation onto Slides for Wholemount FISH
45
TROUBLESHOOTING
53
BrdU Labeling of Chromosomes
57
CONTENTS
58
Analysis of Meiosis of Fixed and Live Oocytes by Light Microscopy
67
GFP and Other Reporters
313
Quantitative Microinjection of Drosophila Embryos
345
GENERAL STRATEGY
346
Quantitative Microinjection of Embryos
353
Targeted Disruption of Gene Function in Drosophila by RNA Interference
361
CONTENTS
362
CONTENTS
375
Generating Antibodies against Drosophila Proteins
389

CONTENTS
68
Cytological Analysis of Spermatocyte Growth and Male Meiosis in Drosophila
87
MethanolAcetone Fixation Technique II
93
PREPARATION OF MEIOTIC CHROMOSOMES
99
Preparation and Analysis of Polytene Chromosomes
111
In Situ Hybridization to Polytene Chromosomes
119
Pretreatment and Hybridization
125
Mapping Protein Distributions on Polytene Chromosomes
131
Preparation of Chromosome Squashes from Thirdinstar Larvae
132
Giemsa Staining
138
Fluorescent Analysis of Drosophila Embryos
141
CONTENTS
143
Imaginal Discs
159
CONTENTS
161
The Neuromuscular Junction
175
Larva and Pupa
201
Dissection Techniques
203
In Situ Hybridization
216
Adult
229
Preparing Thick Sections oe Adult Retinas
236
Preparation of Specimens for Transmission Electron Microscopy
242
Preparation of Thin Sections of Drosophila for Examination by Transmission
245
CHEC
246
Preparing Fixatives for Freezesubstitution
252
hHeptane Permeabilization
259
EXAMPLES
265
Electrophysiological Approaches to the Neuromusculature
273
CONTENTS
274
Functional Assays of the Peripheral and Central Nervous Systems
297
CENTRAL NERVOUS SYSTEM
304
CONTENTS
390
Staining Embryos for Monoclonal Fusion Screening
408
Photoactivated Gene Expression for Cellfate Mapping and Cell Manipulation
413
CONTENTS
414
Inverse PCR
429
ISOLATION DIGESTION AND LIGATION OF GENOMIC DNA
430
OVERVIEW
439
Functional Cell Ablation
449
Antibody Staining of Adult CNS
455
TUNELAntibody Doublelabeling Method for Drosophila Embryos
464
Embryonic Cell Ablation LIsing Lasers
473
Preparation of DNA from coli Cells Containing Bacteriophage
479
0
487
TREATMENT OF MICROARRAYS PRIOR TO HYBRIDIZATION
500
Using Drosophila Genome Databases
510
Preparation and Analysis of Highmolecularweight DNA in Drosophila
525
Culturing Large Populations of Drosophila for Protein Biochemistry
541
CONTENTS
553
Preparation of Membrane Proteins from Drosophila Embryos
563
CONTENTS
564
Embryo Collection and Dechorionation
571
Immunoblotting of Proteins from Single Drosophila Embryos
577
CONTENTS
578
LIFE CYCLE
585
Preparation of Larval and Adult Cuticles for Light Microscopy
601
Exposing Drosophila to Neuroactive Drugs
617
CONTENTS
618
Index
685
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About the author (2000)

Ashbruner is Professor of Biology in the Department of Genetics at the University of Cambridge. By training and inclination, he is a Drosophila geneticist, although for more than a decade, he has not been where he belongs-the lab bench-but in front of computer screens. He spent six years at the European Bioinformatics Institute, first as the Institute's Research Programme Coordinator, and then as its Joint-Head. He is a fellow of the Royal Society and an Honorary Foreign Member of the American Academy of Arts and Sciences.

R. Scott Hawley is a Senior Scientist at the Stowers Institute of Medical Research. Before joining the Stowers Institute in 2001, he was a Professor of Genetics at the University of California at Davis for 10 years. An accomplished textbook author, he has also published over 50 papers and articles in scientific journals. He has also served on the Board of Directors of the Genetics Society of America and is currently on the Editorial Board of "Genetics."

Michelle Walker is an Intellectual Property Specialist for Galileo Laboratories in Santa Clara, California. She earned an MS in Genetics from the University of California at Davis, has written several scientific articles, served as an Editor for the entomological journal "Sociobiology," and spent numerous hours volunteering her time in the field of bioethics and as a scientific mentor for inner-city children.

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