bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assays for Total Protein
Authors: Lovrien Rex , Matulis Daumantas 
Published: June 2004

AbstractThis unit describes three copper‐based assays to quantitate total protein: the biuret method, a variation of the Lowry method (Hartree‐Lowry method), and the bicinchoninic acid (BCA) assay. Acid hydrolysis of a protein is coupled with ninhydrin detection to quantitate amino acid content of a sample. Ultraviolet spectrophotometry is used to measure total protein and evaluate samples for the presence of contaminants. The Coomassie dye binding, or Bradford, assay is a quite simple assay and frequently is quite sensitive, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acid pH. Finally, dry weight measurement is used to quantitate pure protein. Support protocols describe heat sealing glass tubes for acid hydrolysis, sample dialysis in polyacrylamide gel wells to remove low‐molecular‐weight contaminants, and TCA precipitation to precipitate and concentrate proteins and remove low‐molecular‐weight contaminants.
Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
Authors: Zeller Rolf 
Published: June 2004

AbstractThis unit describes selected methods for fixing and sectioning various forms of biological materials ranging from tissues to single cells. Sections prepared according to these protocols can then be used to examine cell and tissue morphology and in studies involving in situ hybridization, immunohistochemistry, and enzyme histochemistry. The basic protocol describes how tissues and embryos can be prepared for sectioning by fixing in paraformaldehyde followed by embedding in wax, while an alternate protocol describes fixation of suspended or cultured cells. Two support protocols cover preserving and fixing organs by perfusion of whole animals with paraformaldehyde and a procedure for sectioning wax blocks of fixed tissue, plus the subsequent mounting of sections onto prepared or “subbed” glass slides.
In Situ Hybridization to Cellular RNA
Authors: Zeller Rolf , Rogers Melissa , Haramis Anna G. , Carrasceo Andrés E. 
Published: June 2004

AbstractIn situ hybridization to cellular RNA is used to determine the cellular localization of specific messages within complex cell populations and tissues. Tissues may either be embedded in paraffin and sectioned on a microtome (seeHybridization Using Paraffin Sections and Cells), or frozen and sectioned in a cryostat (seeHybridization Using Cryosections). RNA contained in the specimens is hybridized to a specific radiolabeled probe (seeSynthesis of35S‐Labled RiboprobesandSynthesis of35S‐Labled Double‐Stranded DNA Probes), which is then detected using film autoradiography or emulsion autoradiography (Chapter 8).
Rapid Solution Application Methods
Authors: Tang Cha‐Min 
Published: June 2004

AbstractSolution changes to deliver solutes of different compositions are required in virtually every cellular electrophysiological experiment. Also, in many neurobiology experiments, it is necessary to make rapid step changes in the concentration of a test compound in order to outpace receptor desensitization or to mimic the brief lifetime of a fast synaptic response. The goal of this unit is to aid the investigator in choosing the rapid solution application method that is most appropriate for the experimental situation and to describe the methods for fabricating two relatively simple devices for making rapid changes between different solutions.
cDNA Library Construction from Single Cells
Authors: Kramer Phillip R. 
Published: June 2004

AbstractcDNA Library Construction from Single Cells (Phillip R. Kramer, National Institute of Neuronal Disorders and Stroke, NIH, Bethesda, Maryland). Analysis of gene expression or comparing gene expression patterns between cells and/or tissues facilitates the identification of molecules activated by a cellular program or by a particular physiological or pharmacological treatment. This unit presents a protocol for studying these molecules at the level of a single cell.
Targeted Toxins
Authors: Wiley Ronald G. , Lappi Douglas A. 
Published: June 2004

AbstractMolecular neurosurgery can be used to make selective neural lesions by targeting cytotoxins to specific populations of neurons based on their common expression of a particular surface molecule. The targeted toxins employed in this unit consist of a targeting moiety (vector) and an effector moiety (cytotoxin). In all cases discussed in this unit, the cytotoxic moiety is an enzyme that catalytically inactivates the large ribosomal subunit, irreversibly inhibiting protein synthesis and resulting in cell death. These toxins appear to kill in an all‐or‐none fashion, indicating that one molecule of free cytotoxin in the cytoplasm of a cell is sufficient to kill the cell. Three general molecular neurosurgery protocols are presented in this unit. The first describes suicide transport, which refers to the use of targeted toxins to make anatomically restricted lesions based on retrograde axonal transport of the toxin. The second involves immunolesioning and uses anti‐neuronal immunotoxins to make type‐selective and anatomically restricted lesions. The final protocol uses neuropeptide‐toxin conjugates to selectively destroy neurons expressing the receptor for the specific neuropeptide.
Cellular Compartment Analysis of Temporal Activity by Fluorescence In Situ Hybridization (catFISH)
Authors: Guzowski John F. , Worley Paul F. 
Published: June 2004

AbstractThis protocol describes a method (cellular compartment analysis of temporal activity by fluorescent in situ hybridization or catFISH) that uses fluorescent in situ hybridization to immediate‐early gene RNAs and confocal microscopy to identify neuronal populations activated at two distinct times. The combination of cellular and temporal resolution makes catFISH a valuable tool for investigating the dynamic interactions of neuronal populations associated with different behaviors or cognitive challenges.
Detection of DNA Damage in Tissue Sections by In Situ Nick Translation
Authors: Chesselet Mary‐Franciose , MacKenzie Larami , Hoang Tuan 
Published: June 2004

AbstractThe technique of in situ nick translation (ISNT) is used to detect DNA strand breaks in tissue sections at the cellular level with great sensitivity. In fact, ISNT can be used to detect DNA damage in a single cell, which is particularly useful to assess programmed cell death during development. One crucial advantage of ISNT is the anatomical resolution that permits a detailed topographical analysis of DNA damage. This can be useful to identify cells that are more vulnerable to an experimental insult. Furthermore, cells with DNA damage can be identified morphologically with this method. This can be of interest to determine whether cells that exhibit DNA damage already exhibit clear features of dying cells or are still relatively intact morphologically. This can be useful to identify the mode of cell death involved. This unit provides a protocol that describes tissue preparation, in situ nick translation and emulsion autoradiography.
Theory and Application of Fluorescence Microscopy
Authors: Coling Donald , Kachar Bechara 
Published: June 2004

AbstractThe growing importance in biology and especially in neurobiology of fluorescence microscopy is due to (1) the extraordinary development of new fluorescent molecular probes and (2) the development of improved low light level imaging systems and confocal microscopy techniques. This overview covers fluorescent molecular probes, filters and filter sets, multiband filters and multidye fluorescence, light sources, microscope objectives, image resolution and the point‐spread function, and general steps for immunolabeling.
Imaging Nervous System Activity
Authors: Fields R. Douglas , O'Donovan Michael J. 
Published: June 2004

AbstractOptical imaging methods rely upon visualization of three types of signals: (1) intrinsic optical signals, including light scattering and reflectance, birefringence, and spectroscopic changes of intrinsic molecules, such as NADH or oxyhemoglobin; (2) changes in fluorescence or absorbance of voltage‐sensitive membrane dyes; and (3) changes in fluorescence or absorbance of calcium‐sensitive indicator dyes. Of these, the most widely used approach is fluorescent microscopy of calcium‐sensitive dyes. This unit describes protocols for the use of calcium‐sensitive dyes and voltage‐dependent dyes for studies of neuronal activity in culture, tissue slices, and en‐bloc preparations of the central nervous system.
Use of FM1‐43 and Other Derivatives to Investigate Neuronal Function
Authors: Cousin Mike 
Published: June 2004

AbstractThis unit describes how to use FM1‐43 and its derivatives to investigate the physiology of synaptic vesicle recycling in neurons. This includes basic protocols for visualizing synaptic vesicle exocytosis and endocytosis and information on more advanced uses of the probes to examine different aspects of the vesicle life cycle.
Identification of Neural Programmed Cell Death Through the Detection of DNA Fragmentation In Situ and by PCR
Authors: Chun Jerold , Blaschke Anne J. 
Published: June 2004

AbstractA universal feature in the development of multicellular organisms is a physiological form of cell death called programmed cell death (PCD). A subset of PCD is apoptosis, which is defined by characteristic morphological changes and genomic DNA fragmentation producing what are referred to as nucleosomal ladders. To understand how PCD operates in a developing tissue or in a tissue following an experimental procedure, dying cells must be identified in relation to their surviving neighbors. One way to accomplish this is to visualize fragmented DNA in situ, in conjunction with gel electrophoresis of isolated DNA to visualize the nucleosomal ladders associated with apoptosis. Two approaches are presented in this unit: in situ end‐labeling plus (ISEL+), a technique to identify dying cells in tissue sections or cell cultures of central nervous system (CNS) tissue (optimized for embryonic samples); and the use of ligation‐mediated polymerase chain reaction (LMPCR) to identify nucleosomal ladders from intact tissues. Also included are procedures for preparing thymocyte cell cultures for use as controls in the ISEL+ procedure and for isolating genomic DNA for LMPCR.
Quantitative Analysis of In Vivo Cell Proliferation
Authors: Cameron Heather A. 
Published: June 2004

AbstractTwo similar techniques, [3H]thymidine autoradiography and 5‐bromo‐2'‐deoxyuridine (BrdU) immunohistochemistry, have been developed to label dividing cells and identify them following survival times of up to several years, so that different aspects of their fate can be examined. Both techniques use markers that are incorporated into the DNA of proliferating cells as it is synthesized during the S phase of mitosis. The techniques differ in the markers used and the detection systems used to recognize them. In the first method described in this unit, tritiated thymidine is injected into animals and is incorporated into dividing cells. These cells can then be identified using emulsion autoradiography. In the second method presented, BrdU, a halogenated thymidine analog, is similarly injected and incorporated into cells in S phase, and then detected immunohistologically with an antibody specific for DNA containing BrdU.
Isolation and Generation of Oligodendrocytes by Immunopanning
Authors: Mayer‐Pröschel Margot 
Published: June 2004

AbstractThis unit presents a procedure for the purification of oligodendrocyte progenitor cells, their expansion in vitro, and differentiation of these cells to yield oligodendrocyte cultures. A variation is also presented, detailing the direct isolation of differentiated oligodendrocytes from postnatal brain. The purification of the target cell population is achieved by exploiting the differential binding of cells to tissue culture dishes coated with an antibody directed against a specific cell‐surface antigen. Cells expressing this surface antigen are retained on the dish and are thereby separated from the remaining cell population.
Generation and Utilization of Phosphorylation State–Specific Antibodies to Investigate Signaling Pathways
Authors: Alberta John A. , Segal Rosalind A. 
Published: June 2004

AbstractPhosphorylation state‐specific antibodies can be of great use, for example, in studying individual steps within a given signal transduction pathway. This unit presents a general approach to the generation and purification of phosphorylation state‐specific antibodies. In addition to their ability to detect phosphorylation at a particular key site, these antibodies are often more sensitive for biochemical studies. Besides their application in immunoblotting procedures, activation state‐specific antibodies can be used as immunohistochemical reagents. Thus, critical changes in phosphorylation can be monitored as described on an individual cell basis or in fixed tissue sections. Such antibodies can be used to address fundamental questions about signal transduction pathways during physiologic events that cannot be resolved by more conventional methodologies.
Homology Cloning of cDNA or Genomic DNA
Authors: Albert Paul R. 
Published: June 2004

AbstractThe principle of cloning utilizes hybridization of single‐stranded DNA probes to denatured DNA fixed on membranes to detect the identical DNA molecules present in a large population of diverse DNAs. In homology or low‐stringency cloning, hybridization of oligonucleotide, cDNA, or genomic DNA probes to denatured DNA from plasmid or bacteriophage libraries is carried out under low‐stringency conditions that promote hybridization in the presence of sequence mismatch (i.e., mispaired nucleotides). The procedures used are identical to those for screening libraries at high stringency, with the exception that hybridization and washing conditions are altered to permit hybridization with mismatched sequence; salt concentration is increased and the hybridization and wash temperatures are decreased. This unit describes conditions that have been used to clone and identify novel genes and cDNA clones using low‐stringency hybridization of known probes to membranes that contain libraries of bacterial or bacteriophage DNA.
Expression Cloning of Neural Genes Using Xenopus laevis Oocytes
Authors: Boulter Jim , Boyer Christopher 
Published: June 2004

AbstractExpression cloning requires a representative cDNA or genomic DNA library and a host organism in which the cloned genes can be transcribed and/or translated. It likewise requires a method to detect the expressed protein using, for example, the inherent biological activity of the gene or antibodies specific for the gene product. Most successful expression cloning strategies have employed cDNA libraries constructed in plasmid or bacteriophage lambda vectors and Xenopus oocytes or cultured mammalian cells as hosts. This unit presents several protocols designed for expression cloning paradigms that rely on electrophysiological recordings from Xenopus laevis oocytes.
Mutagenesis Approaches for Elucidation of Protein Structure‐Function Relationships
Authors: Strader Catherine D. 
Published: June 2004

AbstractSeveral mutagenesis strategies have been used to allow the identification of specific receptor‐ligand interactions to support the docking of ligands within various receptor models. The strengths and limitations of each strategy are discussed in this overview. Large‐scale mapping is described using deletion and chimeric receptors. Fine tuning with single residue replacements is also covered along with two‐dimensional approaches.
Oligonucleotide‐Directed Mutagenesis Without Phenotypic Selection
Authors: Kunkel Thomas A. 
Published: June 2004

AbstractA DNA sequence can be specifically altered by synthesizing the desired sequence change within an oligonucleotide, and then converting this into a biologically active circular DNA strand by using the oligonucleotide to prime in vitro synthesis on a single‐stranded circular template. This unit presents a protocol which uses a DNA template containing a small number of uracil residues in place of thymine. Use of the uracil‐containing template allows rapid and efficient recovery of mutants; in principle this same template can be applied to most of the other mutagenesis protocols in use. The length of the oligonucleotide primer is highly variable and depends on the nature of the change being made.
Directed Mutagenesis Using the Polymerase Chain Reaction
Authors: Cormack Brendan 
Published: June 2004

AbstractThe polymerase chain reaction (PCR) is most often used for the enzymatic amplification and direct sequencing of small quantities of nucleic acids. This technology can also be used as a quick and efficient method for introducing any desired sequence change into the DNA of interest. This unit contains two basic protocols for introducing base changes into specific DNA sequences. The first describes the incorporation of a restriction site and the second details the generation of specific point mutations. An alternate protocol describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.
Overview of the Baculovirus Expression System
Authors: Murphy Cheryl Isaac , Piwnica‐Worms Helen 
Published: June 2004

AbstractBaculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This unit gives an overview of the baculovirus expression system, including discussion of the baculovirus life cycle, and post‐translational modifications that occur in insect cells. In addition, the steps for overproducing proteins in the baculovirus systems are described along with recommendations for choosing an appropriate baculovirus vector and DNA, and reagents and equipment necessary for implementing the whole overexpression system.
Protein Expression in the Baculovirus System
Authors: Bernard Alain , Payton Mark , Radford Kathryn R. 
Published: June 2004

AbstractInsect cell‐recombinant baculovirus co‐cultures offer a protein production system that complements microbial systems by providing recombinant proteins in soluble form and with most post‐translational modifications. Moreover, the large size of the viral genome enables cloning of large segments of DNA and consequent expression of complex protein aggregates. This unit describes methods associated with the large‐scale production of recombinant proteins in the baculovirus expression system. A method for large‐scale production of viral stocks is described and methods for titration of virus are provided (a plaque assay and an end‐point assay). Once viral stocks have been prepared and titered, a protocol for testing the virus in small‐scale cultures is provided to determine the kinetics of expression, which allows evaluation of various cell culture and infection conditions aimed at developing optimal levels of protein production (e.g., comparisons of different host cell lines, media, and environmental parameters). Support protocols provide instructions for preparing culture samples for protein analysis by SDS‐PAGE and discuss analytical methods for monitoring nutrient levels in cell culture fluids. Once optimal process parameters are identified, protocols describe production of the target protein on a large scale in fermentors using either regular batch production in bioreactors or a fed‐batch procedure of production in perfusion cultures. Techniques for harvesting cultures from bioreactors are also provided
Gene Transfer into Neural Cells In Vitro Using Adenoviral Vectors
Authors: Southgate Thomas D. , Kingston Paul A. , Castro Maria G. 
Published: June 2004

AbstractAdenoviruses (Ads) have become a very attractive and versatile vector system for delivering genes into brain cells in vitro and in vivo. One of the main attractions of Ads is that they can mediate gene transfer into post‐mitotic cells, i.e. neurons. Ads are easy to grow and manipulate, stable, and their biology is very well understood. This unit is designed to help newcomers into the field, to design, prepare and grow replication‐defective recombinant adenovirus vectors with the aim of transferring genes into neurons and glial cells in primary culture. It provides step‐by‐step methods describing the preparation of brain cell cultures, their infection using recombinant adenovirus vectors and also the assessment of transgene expression using a variety of techniques including fluorescence immunocytochemistry and fluorescence activated cell‐sorting (FACS) analysis. The methods described will be useful to scientists wishing to enter the adenovirus field to construct adenovirus vectors to be used for gene transfer into neural cells.
Preparation of mRNA for Expression Monitoring
Authors: Byrne Michael C. , Whitley Maryann Z. , Follettie Maximillian T. 
Published: June 2004

AbstractThe ability to construct comprehensive gene expression profiles comprising hundreds to thousands of genes whose RNA levels are monitored simultaneously represents an exciting new capability in molecular biology. This is accomplished by hybridizing mRNA, which has been quantitatively amplified and labeled with biotin, to DNA chips that display thousands of nucleotides complementary to the mRNAs of interest. In this unit, rationale for starting with poly(A+) versus total RNA is discussed, and strategies for choosing oligonucleotides for chip design is presented. Protocols on RNA amplification and labeling, and purifying and quantifying the cDNA and in vitro transcription products are included.
Gene Expression Analysis Using cDNA Microarrays
Authors: Karsten StanisLav L. , Geshwind Daniel H. 
Published: June 2004

AbstractThis unit focuses on cDNA microarrays, which are being increasingly applied to large‐scale gene expression studies in the nervous system, concentrating on the steps of probe preparation and microarray hybridization. Four protocols for the attachment of fluorophores to cDNA (labeling) and cDNA hybridization onto microarrays are presented: direct labeling using either Klenow fragment or reverse transcriptase, and two signal amplification techniques represented by the tyramide signal amplification method (TSA), and detection and labeling of cDNA using PCR amplification. Basic issues in experimental design, sources of variability and potential pitfalls in microarray experiments are described. Anticipated results and follow up of microarray experiments, including confirmation of the results and troubleshooting are also discussed.
Overview of Gene Targeting by Homologous Recombination
Authors: Mortensen Richard 
Published: June 2004

AbstractOverview of Gene Targeting by Homologous Recombination (Richard Mortensen, University of Michigan Medical School, Ann Arbor, Michigan).This overview describes the basic methodology and applications of homologous recombination. Both insertion and replacement constructs are discussed along with methods of selection. Gene mutation and inactivation to produce a knockout animal can be accomplished in several ways, and the Cre‐loxP system is described for this purpose in some detail.