A Specialized Method to Resolve Fine 3D Features of Astrocytes in Nonhuman Primate (Marmoset, Callithrix jacchus) and Human Fixed Brain Samples.

in Methods in Molecular Biology (2019), 1938

Astrocytes are among the most numerous cells in the brain and fulfill diverse functions in homeostasis and regulation of neuronal activity. Astrocytes also dramatically change their properties in response ... [more ▼]

Astrocytes are among the most numerous cells in the brain and fulfill diverse functions in homeostasis and regulation of neuronal activity. Astrocytes also dramatically change their properties in response to brain injury or disease, a process called reactive gliosis. Precisely how astrocytes contribute to healthy brain function and play differential roles in brain pathology and regeneration remain important areas of investigation. To better understand the properties of astrocytes, more sophisticated approaches for probing their rich and complex anatomical and molecular features are needed to fully determine their contribution to brain physiology. Here we present an efficient and straightforward immunolabeling protocol to obtain high-resolution fluorescence-based images from fixed nonhuman primate (common marmoset Callithrix jacchus) and human brain samples. Importantly, the protocol is useful for obtaining images from samples that have been stored in fixative solutions (such as formalin) for years. This approach is especially useful for three-dimensional, multichannel confocal microscopy and can be optimized for super-resolution techniques such as stimulated emission depletion (STED) microscopy. We also present a strategy for using specific combinations of markers to define the phenotypic variations and cellular/subcellular properties of astrocytes to better predict the function of these cells on their surrounding brain microenvironment. [less ▲]

Resources for Systems Genetics.

in Methods in Molecular Biology (2017), 1488

A key characteristic of systems genetics is its reliance on populations that vary to a greater or lesser degree in genetic complexity-from highly admixed populations such as the Collaborative Cross and ... [more ▼]

A key characteristic of systems genetics is its reliance on populations that vary to a greater or lesser degree in genetic complexity-from highly admixed populations such as the Collaborative Cross and Diversity Outcross to relatively simple crosses such as sets of consomic strains and reduced complexity crosses. This protocol is intended to help investigators make more informed decisions about choices of resources given different types of questions. We consider factors such as costs, availability, and ease of breeding for common scenarios. In general, we recommend using complementary resources and minimizing depth of resampling of any given genome or strain. [less ▲]

A Guide to Integrating Transcriptional Regulatory and Metabolic Networks Using PROM (Probabilistic Regulation of Metabolism)

in Methods in Molecular Biology (2013), 985

The integration of transcriptional regulatory and metabolic networks is a crucial step in the process of predicting metabolic behaviors that emerge from either genetic or environmental changes. Here, we ... [more ▼]

The integration of transcriptional regulatory and metabolic networks is a crucial step in the process of predicting metabolic behaviors that emerge from either genetic or environmental changes. Here, we present a guide to PROM (probabilistic regulation of metabolism), an automated method for the construction and simulation of integrated metabolic and transcriptional regulatory networks that enables large-scale phenotypic predictions for a wide range of model organisms. [less ▲]

Detection of activated STAT species using electrophoretic mobility shift assay (EMSA) and potential pitfalls arising from the use of detergents.

in Methods in Molecular Biology (2013), 967

Here we describe the preparation of nuclear extracts and the electrophoretic mobility shift assay (EMSA) for the detection of STAT species. We use the method for the investigation of STAT1 and STAT3 homo ... [more ▼]

Here we describe the preparation of nuclear extracts and the electrophoretic mobility shift assay (EMSA) for the detection of STAT species. We use the method for the investigation of STAT1 and STAT3 homo- and heterodimers and show how the preparation of the extracts can influence the distribution of the STAT species observed in the EMSA. We show that detergents can massively influence the STAT dimer distribution. Although it is unclear whether they primarily interfere with STAT DNA binding and/or whether they break up or further oligomerize STATs, the observation may also have an impact on the results of other techniques performed with detergent-containing cell lysates (e.g., coimmunoprecipitations of STATs with other proteins). [less ▲]

Integration of proteomic and metabolomic profiling as well as metabolic modeling for the functional analysis of metabolic networks.

in Methods in Molecular Biology (2011), 694

The integrated analysis of different omics-level data sets is most naturally performed in the context of common process or pathway association. In this chapter, the two basic approaches for a metabolic ... [more ▼]

The integrated analysis of different omics-level data sets is most naturally performed in the context of common process or pathway association. In this chapter, the two basic approaches for a metabolic pathway-centric integration of proteomics and metabolomics data are described: the knowledge-based approach relying on existing metabolic pathway information, and a data-driven approach that aims to deduce functional (pathway) associations directly from the data. Relevant algorithmic approaches for the generation of metabolic networks of model organisms, their functional analysis, database resources, visualization and analysis tools will be described. The use of proteomics data in the process of metabolic network reconstruction will be discussed. [less ▲]