In Situ Hybridization Protocols for the Brain

by Wisden; Morris

• ISBN13: 9780123668479
• ISBN10: 0123668476
• Edition: 2nd
• Format: Hardcover
• Copyright: 2002-08-05
• Publisher: Elsevier Science

Summary

This volume of the International Review of Neurobiology was written to assist researchers without any previous experience with in situ hybridization, allowing them to follow the protocols and expect good results. It contains all the information required for newcomers to achieve successful in situ hybridization results, and methods for improving the technique of those already utilizing it. Published since 1959, International Review of Neurobiology is a well-known series appealing to neuroscientists, clinicians, psychologists, physiologists, and pharmacologists. Led by an internationally renowned editorial board, this important serial publishes both eclectic volumes made up of timely reviews and thematic volumes that focus on recent progress in a specific area of neurobiology research. Published since 1959, International Review of Neurobiology is a well-known series appealing to neuroscientists, clinicians, psychologists, physiologists, and pharmacologists. Led by an internationally renowned editorial board, this important serial publishes both eclectic volumes made up of timely reviews and thematic volumes that focus on recent progress in a specific area of neurobiology research.

Table of Contents

Contributors	xi
Acknowledgements	xiii
How to Use This Book	xv
Introduction	xvii
Introduction: Studying Gene Expression in Neural Tissues by In Situ Hybridization
W. Wisden
B. J. Morris
PART I In Situ Hybridization with Radiolabelled Oligonucleotides
In Situ Hybridization with Oligonucleotide Probes
W. Wisden
B. J. Morris
Introduction: mRNA Hybridization Using Oligodeoxyribonucleotide Probes	4	(1)
ISH with Oligonucleotides	4	(2)
Advantages of Using Oligonucleotides for ISH	6	(5)
Problems	11	(1)
Preparation of Sections	11	(4)
Oligonucleotide Probes: Synthesis and Design	15	(6)
Probe Labelling with Terminal Deoxynucleotidyl Transferase (TdT)	21	(11)
Hybridization and Washing	32	(6)
Exposure to X-ray Film and Dipping in Emulsion	38	(10)
Controls for ISH	48	(2)
Artefacts and Failures: Troubleshooting	50	(3)
Presentation of Autoradiographs	53	(8)
Acknowledgements	56	(1)
References	56	(5)
Cryostat Sectioning of Brains
V. Revilla
A. Jones
Introduction	61	(1)
Parts of a Cryostat	61	(1)
Tissues	62	(1)
Mounting Brain on Specimen Disc	63	(1)
Sectioning	63	(2)
Troubleshooting Guide	65	(6)
Processing Rodent Embryonic and Early Postnatal Tissue for In Situ Hybridization with Radiolabelled Oligonucleotides
D. J. Laurie
P. C. U. Schrotz
H. Monyer
U. Amtmann
Introduction	71	(1)
Standardizing Animal Age	72	(3)
Tissue Excision	75	(2)
Sectioning Tips	77	(3)
Hybridization Tips	80	(1)
Structure Identification	81	(4)
Acknowledgements	81	(1)
References	81	(4)
Processing Retinal Tissue for In Situ Hybridization
F. Muller
Introduction	85	(2)
Anatomy of the Retina	87	(2)
Preparation of the Retina	89	(2)
Sectioning and In Situ Hybridization	91	(2)
References	92	(1)
Processing the Spinal Cord for In Situ Hybridization with Radiolabelled Oligonucleotides
A. Berthele
T. R. Tolle
Introduction	93	(1)
Spinal Cord Preparation	94	(1)
Spinal Cord Sectioning	95	(5)
Hybridization	100	(1)
Quantification	101	(2)
References	102	(1)
Processing Human Brain Tissue for In Situ Hybridization with Radiolabelled Oligonucleotides
L. F. B. Nicholson
Introduction	103	(4)
Collection of Tissue	107	(2)
Cryostat Sectioning	109	(3)
Procedures for Using Radioactive Oligonucleotide Probes	112	(3)
Acknowledgements	113	(1)
References	113	(2)
In Situ Hybridization of Astrocytes and Neurons Cultured In Vitro
L. Ariza-McNaughton
C. De Felipe
S. P. Hunt
Introduction	115	(1)
Hybridization Procedures for Lab-Tek Tissue Culture Chamber/Slides	116	(2)
Hybridization Procedures for Glass Coverslips	118	(5)
References	122	(1)
In Situ Hybridization on Organotypic Slice Cultures
A. Gerfin-Moser
H. Monyer
Introduction	123	(5)
Preparation of Slice Cultures	128	(1)
In Situ Hybridization	129	(2)
Autoradiography	131	(2)
Acknowledgements	132	(1)
References	132	(1)
Quantitative Analysis of In Situ Hybridization Histochemistry
A. L. Gundlach
R. D. O'Shea
Introduction	133	(2)
General Considerations	135	(3)
Computerized Image Analysis Systems	138	(2)
Quantification of Film Images of ISH	140	(12)
Quantification of Nuclear Emulsion Images of ISH	152	(9)
Quantification of Non-isotopic ISH	161	(2)
Statistical Analysis and Reporting of Results	163	(2)
Reflections and Future Directions	165	(1)
Conclusion	166	(5)
Acknowledgements	166	(1)
References	166	(5)
PART II Non-radioactive In Situ Hybridization
Non-radioactive In Situ Hybridization Using Alkaline Phosphatase-labelled Oligonucleotides
S. J. Augood
E. M. McGowan
B. R. Finsen
B. Heppelmann
P. C. Emson
Introduction to Alkaline Phosphatase-labelled Oligonucleotides	171	(2)
Preparation of Fresh Frozen CNS Tissue Sections	173	(5)
Hybridization	178	(3)
Post-hybridization Washing and Colour Development	181	(4)
Use of AP-oligonucleotides for Semiquantitative Analysis	185	(6)
Suitable Controls for ISH	191	(1)
Combining AP-oligonucleotides and [35S]Oligonucleotides: Co-expression Studies	191	(3)
Combining AP ISH and Immunocytochemistry	194	(2)
Troubleshooting	196	(6)
Acknowledgments	196	(1)
References	197	(5)
Combining Non-radioactive In Situ Hybridization with Immunohistological and Anatomical Techniques
P. Wahle
Introduction	202	(2)
The Probes	204	(6)
The Tissue	210	(5)
Troubleshooting Tissue Quality	215	(1)
The In Situ Hybridization	215	(11)
Combining Techniques: Immunohistochemistry, Tract Tracing, Triple Labelling	226	(7)
Analysis and Presentation	233	(4)
Acknowledgements	235	(1)
References	235	(2)
Non-radioactive In Situ Hybridization: Simplified Procedures for Use in Whole-mounts of Mouse and Chick Embryos
L. Ariza-McNaughton
R. Krumlauf
Introduction	237	(2)
Improved Whole-mount Methodology	239	(1)
Probe Synthesis	239	(1)
Preparation of Tissue	239	(4)
Hybridization	243	(1)
Processing for Histology	243	(3)
Notes	246	(1)
Detailed Recipes	246	(3)
References	247	(2)
Index	249