Neurotrophin Protocols, 2001 Methods in Molecular Biology Series, Vol. 169

The past decade has seen an extraordinary growth in research interest in neurotrophic factors, and the study of the neurotrophin family has led this activity. Nevertheless, this area of research has often struggled as a result of techniques that were either inadequate or just emerging from other research fields and disciplines. Neurotrophin Protocols has brought together many leaders in the neurotrophin field who detail their special expertise in a wide variety of techniques. Though most procedures are valid across many diff- ent fields of research, some of those described here have been developed to address particular issues within the neurotrophic factor field. The protocols cover a broad range of biochemical, histological, and biological techniques that are often required by the modern laboratory. However, all have been written with sufficient detail to allow any laboratory to achieve proficiency without need of reference to other texts. Neurotrophin Protocols is divided into four sections dealing with p- tein, RNA, recombinant, and in vivo techniques. Protein techniques have in general been less successfully employed than those dealing with RNA or DNA. However, procedures that achieve localization and quantification of the neurotrophins are now being used more extensively. Their inclusion here should assist further studies at the protein level. Transgenic cell lines and animals are commonplace in the scientific research literature, but their inc- sion in several chapters in this book provide some novel uses that are not readily available elsewhere.

Neurotrophin Quantification and Localization.- Localization of Neurotrophin Proteins Within the Central Nervous System by Immunohistochemistry.- Neurotrophin Immunohistochemistry in Peripheral Tissues.- Extraction and Quantification of the Neurotrophins.- Identifying Novel Proteins in Nervous Tissue Using Microsequencing Techniques.- RNA Quantification and Localization.- Quantification of mRNA Levels Using Northern Blotting.- Quantification of mRNA Levels Using Ribonuclease Protection Assay.- Quantification of Neurotrophin mRNA by RT-PCR.- Sensitive and Nonradioactive In Situ Detection of Neurotrophin mRNAs in the Nervous System.- Construction of Recombinant Proteins,and Transgenic Cells and Animals.- Expression of Neurotrophic Factors such as GDNF in the E. coli System.- Construction of Cells Expressing Neurotrophins.- Efficient Generation of Stable Pheochromocytoma (PC12) Cell Lines Using a Recombinant Retrovirus (LNC).- Construction and Analysis of Transgenic Animals Overexpressing Neurotrophins.- Construction of Transgenic Animals Overproducing Neurotrophins and Their Receptors.- In Vivo Analysis.- Tracing with Radiolabeled Neurotrophins.- Toxin-Induced Death of Neurotrophin-Sensitive Neurons.- Oligonucleotides as Inhibitors of Protein Synthesis.- The IODO-GEN Method for Labeling and the Use of Retrograde Axonal Transport to Assay Neurotrophins.- Stereological Estimation of Neuronal Number The Optical Disector/Cavalieri Combination.

Includes supplementary material: sn.pub/extras