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Critical to understanding biological roles for transcription factors is an appreciation of the target genes regulated by the transcription factor. The identification of target genes can often expand the understanding of known biological roles for a transcription factor and may reveal unappreciated and unexpected functions. This article focuses on the identification and characterization of transcription...
The expression of a number of chemokines and chemokine receptors by cells resident in normal and pathological central nervous system (CNS) tissue has been characterized by in situ hybridization techniques. As a result, our understanding of the role of this cytokine family in neurobiology has been enhanced greatly. Specific methods for detecting chemokine and chemokine receptor mRNAs in situ vary with...
Serine protease inhibitors (serpins) constitute a still expanding superfamily of structural similar proteins, which are localized extracellularly and intracellularly. Serpins play a central role in the regulation of a wide variety of (patho) physiological processes including coagulation, fibrinolysis, inflammation, development, tumor invasion, and apoptosis. Serpins have a unique mechanism of inhibition...
Protein antigens administered via the oral route are exposed to a hostile environment in the gastrointestinal tract, consisting of digestive enzymes and a range of pH (1–7.5). Using a delivery system can afford protection to entrapped components against degradation and permit delivery of antigen to the cells responsible for generating local and systemic immune responses. In this comparative study,...
Cell death induction by apoptosis is an important process in the maintenance of tissue homeostasis as well as tissue destruction during various pathological processes. Consequently, detection of apoptotic cells in situ represents an important technique to assess the extent and impact of cell death in the respective tissue. While scoring of apoptosis by histological assessment of apoptotic cells is...
Mitochondria are cytoplasmic, double-membrane organelles, a main role of which is to synthesize ATP, the universal energy ‘supply’ of cells. In the last three decades, molecular genetic, biochemical, immunological and cell biological techniques have been applied in a coordinated fashion to unveil the pathogenesis of known mitochondrial disorders, as well as to explore the role of mitochondria in aging...
An oncolytic virus is considered a targeted cancer therapy due to its ability to specifically target, replicate in and lyse cancer cells while leaving normal cells unharmed. Over the last few years several tumor selective oncolytic viruses have been developed. These include certain DNA viruses such as adenovirus that have been genetically manipulated to target specific cancer cells by exerting restrictions...
This manuscript details a protocol for the co-localization of a microRNA and its putative protein target in paraffin embedded formalin fixed tissues. The key variables for the first step, microRNA in situ hybridization, includes probe concentration (1–2pmol/μl), locked nucleic acid (LNA) modified probes, protease digestion (pepsin 1.3mg/ml), and a low stringency wash. Key variables for the subsequent...
Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and...
This manuscript focuses on the detection of viral nucleic acids by in situ based methodologies. The optimal protocol depends on the virus. We will describe protocols for viral RNA detection by reverse transcriptase (RT) in situ PCR. We will also directly compare this method to the detection of viral RNA using standard in situ hybridization with locked nucleic acid (LNA) probes. Most DNA viruses are...
After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin...
In recent years the Drosophila heart has become an established model for many different aspects of human cardiac disease. This model has allowed identification of disease-causing mechanisms underlying congenital heart disease and cardiomyopathies and has permitted the study of underlying genetic, metabolic and age-related contributions to heart function. In this review we discuss methods currently...
To achieve specificity and sensitivity using immunohistochemistry it is necessary to combine the application of validated primary antibodies with optimised pre-treatment, detection and visualisation steps. The influence of these surrounding procedures is reviewed. A practical evaluation of tyramide signal amplification and rolling circle amplification detection methods is provided in which formalin...
As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for “research use” continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an...
Genetic methods of antibody generation offer a highly tuneable tool for the production of target specific reagents suitable for a wide range of applications, including immunohistochemistry. The direct linkage between binder phenotype and genotype enables the rapid identification and manipulation of specific binders into formats suitable for highly specific and sensitive detection of targets from soluble...
PTEN is an important tumor suppressor gene. Interpreting PTEN deficiency in the appropriate microscopic context of cancer may be important to understand its role in tumor development and progression. This may be particularly relevant in heterogeneous tumors. Here, we discuss the usefulness of 3D cultures in understanding the consequences of PTEN inactivation in tissue architecture. Afterwards, we...
Pten encodes a well-characterized protein that is important in several cancers due to its tumor suppressor function. Yet, the detection and evaluation of PTEN by immunohistochemistry (IHC) for clinical practice have not been standardized. Thus, in this study, we performed a literature review of protocols for PTEN assessment by IHC and the possible differences in evaluation, based on our experience...
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