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Author: Ingrid Schmid

Corresponding author ([email protected])

INTRODUCTION

Flow cytometry is frequently used to assess nucleic acid content in individual cells. Based on DNA content alone, however, cells in the quiescent G0 phase cannot be discriminated from cells in the proliferative G1 phase, as DNA content remains constant until S-phase entry. In contrast, by measuring RNA content in addition to DNA content, cells can be assigned to G0 and cell-cycle subcompartments of G1. Assessing phenotype at the same time as nucleic acid content allows determination of the cell-cycle status of subpopulations in mixed-cell preparations. This protocol describes an optimized method for combining dual-color cell-surface immunofluorescent staining with staining for DNA-RNA, adapted for a basic dual-laser flow cytometer with blue (488-nm) and red (633- or 647-nm) excitation. DNA is stained at low pH in the presence of saponin with 7-aminoactinomycin D (7-AAD), and RNA is stained wi

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Authors: Anna R. Mäkelä, Wolfgang Ernst, Reingard Grabherr and Christian Oker-Blom

Adapted from Gene Transfer: Delivery and Expression of DNA and RNA (ed. Friedmann and Rossi). CSHL Press, Cold Spring Harbor, NY, USA, 2007.

INTRODUCTION

The baculovirus expression vector system has been used extensively to produce numerous proteins originating from both prokaryotic and eukaryotic sources. In addition to easy cloning techniques and abundant viral propagation, the system’s insect cell environment provides eukaryotic post-translational modification machinery. The baculovirus display vector system provides a number of advantages over prokaryotic systems, allowing the combination of genotype with phenotype, enabling presentation of foreign peptides or even complex proteins on the baculoviral envelope or capsid. Baculoviruses permit larger gene insertions, are easily propagated, and can be grown to high titers. Furthermore, surface modifications of the viral

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Authors: Timothy Walston and Jeff Hardin

Adapted from Imaging in Developmental Biology (ed. Sharpe and Wong). CSHL Press, Cold Spring Harbor, NY, USA, 2011 (in press).

INTRODUCTION

The Caenorhabditis elegans embryo is particularly amenable to microscopy and embryological studies because of its short developmental time, transparent shell, and nonpigmented cells. The agar mount described in this protocol is an easy way to prepare live C. elegans embryos for microscopic visualization. The mount slightly embeds the embryo in agar to hold it in place. The mount also slightly compresses the embryo to provide consistent orientation such that every embryo will be positioned with either its right side or its left side facing the objective. Other techniques can result in random orientations that complicate analysis and make identification of individual blastomeres more challenging.

RELATED INFORMATION

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Authors: Kristina D. Micheva, Nancy O’Rourke, Brad Busse and Stephen J. Smith

Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2010.

INTRODUCTION

Array tomography, which is described in this article, is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of stain

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Authors: Andrea E. Granstedt, Bernd Kuhn, Samuel S.-H. Wang and Lynn W. Enquist

Corresponding author ([email protected]).

INTRODUCTION

Pseudorabies virus (PRV) is a neuroinvasive virus of the herpes family that has a broad host range but does not infect higher-order primates. PRV characteristically travels along chains of synaptically connected neurons and has been used extensively for elucidating neural circuits in the peripheral and central nervous system in vivo. The recombinant virus PRV369 is an attenuated retrograde tracer that encodes G-CaMP2, a fluorescent calcium sensor protein that is stable at physiological pH and mammalian temperature. This protocol describes the use of PRV369 to express G-CaMP2 in a neuronal circuit and to monitor its activity in a living animal, specifically in the submandibular ganglia (SMG), the peripheral parasympathetic ganglia that innervate the salivary glands. The procedure describes the delivery of PRV369 to the glands and shows

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Authors: Parizad M. Bilimoria and Azad Bonni1

Corresponding author ([email protected])

INTRODUCTION

Primary cultures of granule neurons from the post-natal rat cerebellum provide an excellent model system for molecular and cell biological studies of neuronal development and function. The cerebellar cortex, with its highly organized structure and few neuronal subtypes, offers a well-characterized neural circuitry. Many fundamental insights into the processes of neuronal apoptosis, migration, and differentiation in the mammalian central nervous system have come from investigating granule neurons in vitro. Granule neurons are the most abundant type of neurons in the brain. In addition to the sheer volume of granule neurons, the homogeneity of the population and the fact that they can be transfected with ease render them ideal for elucidating the molecular basis of neuronal development. This protocol for isolating granule neurons from post-natal rats is relativel

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Authors: Manuel Théry and Matthieu Piel

Corresponding authors ([email protected]; [email protected])

INTRODUCTION

This protocol describes a simple, fast, and efficient method for making adhesive micropatterns that can be used to control individual cell shape and adhesion patterns. It is based on the use of an elastomeric stamp containing microfeatures to print proteins on the substrate of choice. The process can be subdivided into three parts. First, a silicon master is fabricated, which contains the microfeatures of interest. Once fabricated, the master can be used multiple times to make stamps. Masters with customized patterns can also be purchased commercially. Second, a polydimethylsiloxane (PDMS) stamp is fabricated. Unlike fabrication of the master, this step can be performed without specialized equipment. The PDMS stamp is inked with extracellular matrix proteins. Proteins are printed on a substrate (e.g., a tissue culture polystyrene d

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Author: Molecular Profiling Initiative, NCI

This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.

Investigators can utilize X chromosome inactivation (methylation) to determine the clonality status of a tumor or premalignant lesion in females. The technique is based on a methylation-sensitive restriction enzyme and analysis of a polymorphic locus on the X chromosome. Clonal cell populations will show "loss" of the non-methylated allele after restriction digest. The assay can be performed on DNA recovered from microdissected samples. Both frozen tissue and fixed-embedded archival tissue can be utilized.

Materials

1. DNA sample (see Processing of Microdissected Tissue - DNA-based Analysis)

• Proteinase K (Sigma)

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Author: Molecular Profiling Initiative, NCI

This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.

This method is used to detect genomic DNA deletions in tumor cells. For a more detailed discussion of applying this approach to microdissected samples, see Allelic Loss Studies in Prostate MP at NCI.

Reagents

scientificprotocols

Author: Marcus Marvin

Nuclei preparation

Preparing the cells

1. 300–500 ml cultures of density 1.5–4.0 x 10e7 cells/ml

• Fix cells with freshly prepared formaldehyde in Buffer A for 2 minutes, stirring, to a final concentration of 1%
• Quench fixation by adding glycine to final concentration of 0.125 M
• Wash cells twice in Buffer A