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Quantification of Th1 and Th17 Cells with Intracellular Staining Following PMA/Ionomycin Stimulation
Fridrik Karlsson, Mina Hassan‐Zahraee

Cytokine‐producing cells are at the center of the adaptive immune responses, and quantifying these cells is an important aspect to build understanding of the immune response.

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Whole Blood Measurement of Histone Modifications Linked to the Epigenetic Regulation of Gene Expression
Maria Watson, David Hedley

Rapid progress is being made to understand the regulatory mechanisms that underlie the epigenetic control of gene expression through histone modification. It is now recognized that this plays a major role in normal development and disease.

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Application of Click Chemistry Conditions for 5‐Bromo‐2′‐Deoxyuridine Determination Through Fenton and Related Reactions
Paolo Cappella, Maurizio Pulici, Fabio Gasparri

Mixtures of ascorbate and copper used in certain click chemistry experimental conditions act as oxidizing agents, catalyzing the formation of reactive oxygen species through Fenton and related reactions.

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Digital Data Acquisition and Processing
Mark A. Naivar, David W. Galbraith

A flow cytometer is made up of many different subsystems that work together to measure the optical properties of individual cells within a sample.

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Light Sheet Fluorescence Microscopy (LSFM)
Michael W. Adams, Andrew F. Loftus, Sarah E. Dunn, Matthew S. Joens, James A.J. Fitzpatrick

The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane.

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Semi‐Automated Object Tracking Methods in Biological Imaging
Michael A. Davis, Ondřej Pražský, Laura R. Sysko

Time‐lapse imaging is a rich data source offering potential kinetic information of cellular activity and behavior.

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Statistical Methods for Genome‐Wide and Sequencing Association Studies of Complex Traits in Related Samples
Timothy A. Thornton

Genome‐wide association studies (GWAS) and sequencing studies are routinely conducted for the identification of genetic variants that are associated with complex traits. Many genetic studies for association mapping include related individuals.

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Preparation of Cells from Formalin‐Fixed, Paraffin‐Embedded Tissue for Use in Fluorescence In Situ Hybridization (FISH) Experiments
Stanislawa Weremowicz

Numerical and structural chromosome abnormalities can be accurately detected in cells from archived tissues using fluorescence in situ hybridization (FISH). This unit describes two common approaches to performing FISH in formalin‐fixed, paraffin‐embedded tissue.

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Molecular Cytogenetic Analysis of Telomere Rearrangements
Christa Lese Martin, David H. Ledbetter

Genomic imbalances involving the telomeric regions of human chromosomes, which contain the highest gene concentration in the genome, are proposed to have severe phenotypic consequences.

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Noninvasive Prenatal Testing Using Cell‐Free Fetal DNA in Maternal Plasma
Nilesh Dharajiya, Tricia Zwiefelhofer, Xiaojun Guan, Vach Angkachatchai, Juan‐Sebastian Saldivar

Noninvasive prenatal testing (NIPT) represents an outstanding example of how novel scientific discoveries can be quickly and successfully developed into hugely impactful clinical diagnostic tests.

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Stranded Whole Transcriptome RNA‐Seq for All RNA Types
David F.B. Miller, Pearlly X. Yan, Fang Fang, Aaron Buechlein, James B. Ford, Haixu Tang, Tim H. Huang, Matthew E. Burow, Yunlong Liu, Douglas B. Rusch, Kenneth P. Nephew

Stranded whole transcriptome RNA‐Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to, miRNA (microRNA), piRNA (Piwi‐interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non‐coding intergenic RNA), SRP RNA (signal reco.

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Diagnosing Lysosomal Storage Disorders: Mucopolysaccharidosis Type I
Britt A. Johnson, Angela Dajnoki, Olaf A. Bodamer

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha iduronidase (IDUA).

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Using CellProfiler for Automatic Identification and Measurement of Biological Objects in Images
Mark‐Anthony Bray, Martha S. Vokes, Anne E. Carpenter

Visual analysis is required to perform many biological experiments, from counting colonies to measuring the size or fluorescence intensity of individual cells or organisms.

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Parallel Protein Detection by Solid‐Phase Proximity Ligation Assay with Real‐Time PCR or Sequencing
Tonge Ebai, Masood Kamali‐Moghaddam, Ulf Landegren

Proximity ligation assays are a group of protein detection techniques in which reagents with affinity for target proteins, typically antibodies, are coupled to short strands of DNA.

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ATAC‐seq: A Method for Assaying Chromatin Accessibility Genome‐Wide
Jason D. Buenrostro, Beijing Wu, Howard Y. Chang, William J. Greenleaf

This unit describes Assay for Transposase‐Accessible Chromatin with high‐throughput sequencing (ATAC‐seq), a method for mapping chromatin accessibility genome‐wide.

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BioTAP‐XL: Cross‐linking/Tandem Affinity Purification to Study DNA Targets, RNA, and Protein Components of Chromatin‐Associated Complexes
Artyom A. Alekseyenko, Kyle A. McElroy, Hyuckjoon Kang, Barry M. Zee, Peter V. Kharchenko, Mitzi I. Kuroda

In order to understand how chromatin complexes function in the nucleus, it is important to obtain a comprehensive picture of their protein, DNA, and RNA components, as well as their mutual interactions.

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RNA Interference in Caenorhabditis elegans
Darryl Conte, Lesley T. MacNeil, Albertha J.M. Walhout, Craig C. Mello

RNAi has become an essential tool in C. elegans research. This unit describes procedures for RNAi in C.

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Label‐Free Biomedical Imaging of Lipids by Stimulated Raman Scattering Microscopy
Prasanna V. Ramachandran, Ayse Sena Mutlu, Meng C. Wang

Advances in modern optical microscopy have provided unparalleled tools to study intracellular structure and function, yet visualizing lipid molecules within a cell remains challenging.

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Non‐isotopic RNA In Situ Hybridization on Embryonic Sections
Yacine Touahri, Lata Adnani, Pierre Mattar, Kathryn Markham, Natalia Klenin, Carol Schuurmans

This unit describes methods for non‐isotopic RNA in situ hybridization on embryonic mouse sections. These methods can be used to follow the spatiotemporal dynamics of gene expression in an embryonic tissue of interest. They involve the use of labeled (e.g.

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Imaging the Insertion of Superecliptic pHluorin‐Labeled Dopamine D2 Receptor Using Total Internal Reflection Fluorescence Microscopy
Kathryn M. Daly, Yun Li, Da‐Ting Lin

A better understanding of mechanisms governing receptor insertion to the plasma membrane (PM) requires an experimental approach with excellent spatial and temporal resolutions.

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Quantifying Recording Quality in In Vivo Striatal Recordings
Danielle M. Friend, Caleb Kemere, Alexxai V. Kravitz

The striatum mediates a variety of functions including movement, decision‐making, motivation, and reward learning. In vivo recording is a powerful technique that allows for the interrogation of these striatal functions while an animal is awake and behaving.

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Sampling Phasic Dopamine Signaling with Fast‐Scan Cyclic Voltammetry in Awake, Behaving Rats
S.M. Fortin, J.J. Cone, S. Ng‐Evans, J.E. McCutcheon, M.F. Roitman

Fast‐scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the in vivo measurement of extracellular fluctuations in multiple chemical species.

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Avoidance Behavior: A Free‐Operant Lever‐Press Avoidance Task for the Assessment of the Effects of Safety Signals
Anushka B.P. Fernando, Adam C. Mar, Gonzalo P. Urcelay, Anthony Dickinson, Trevor W. Robbins

This protocol details a free‐operant avoidance paradigm that has been developed to evaluate the relative contribution of different sources of reinforcement of avoidance behavior that may play an important role in the development and maintenance of human anxiety disorders.

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To Stress or Not to Stress: A Question of Models
J. Megan Gray, Francis Chaouloff, Matthew N. Hill

Stress research is a rapidly evolving field that encompasses numerous disciplines ranging from neuroscience to metabolism.

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