3/9/2024 0 Comments Fiji imagej edge colorsManual quantification of large numbers of tissue samples is time-intensive and subject to cognitive bias, resulting in variable and erroneous results. Quantification of images produced from these techniques can be performed manually or with the assistance of commercially available image analysis programs. This method is often combined with a nuclear stain and/or a cytoplasmic stain to aid analysis. The PLA uses a combination of antibody-based detection methods coupled to rolling circle DNA synthesis to create fluorescent foci when two target proteins are in close proximity. A new technology, based on antibody-antigen recognition, is the proximity ligation assay (PLA) that detects sub-cellular single molecule protein interactions in fixed cells via high-resolution fluorescence microscope 7. Research applications of DAB+ IHC include defining cellular phenotype within tissues, investigating the spatiotemporal expression patterns of proteins in tissues and investigating how proteins change with exogenous and endogenous stimuli. The DAB+ precipitate is stable and permits antigen identification in tissues in situ through bright-field light microscopy 5, 6. Routine IHC employs the use of thermodynamically and chemically stable 3,3 ′-diaminobenzidine (DAB), which oxidizes in the presence of hydrogen peroxide or peroxidase conjugated antibodies and hemoglobin to produce a dark brown precipitate. For example, the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), proliferation marker protein Ki67 and epidermal growth factor receptor (EGFR) is used for prognostication in patients diagnosed with breast cancer and informs treatment decisions 1, 2, 3, 4. Immunohistochemistry (IHC) is used for the detection and quantification of target antigens in formalin-fixed paraffin embedded tissue sections for a variety of research applications and human cancer phenotyping. Immune-based antigen recognition techniques and quantitative image analysis in cells and tissues are widely used in scientific research. Andy’s Algorithms are available at GitHub, publicly accessed at. Andy’s algorithms provide a simpler, faster, standardized work flow compared to existing programs, while offering equivalent performance and additional features, in a free to use open-source application of FIJI. Andy’s Algorithms incorporates a step-by-step tutorial and optimization pipeline to make batch image analysis simple for the untrained user and adaptable across laboratories. To overcome this, we have designed Andy’s Algorithms, a series of automated image analysis pipelines for FIJI, that permits rapid, accurate and reproducible batch-processing of 3,3 ′-diaminobenzidine (DAB) immunohistochemistry, proximity ligation assays (PLAs) and other common assays. Accurate high-throughput quantitation of these assays using general image analysis software can be time consuming and challenging, particularly when attempted by users with limited image processing and analysis knowledge. Quantification of cellular antigens and their interactions via antibody-based detection methods are widely used in scientific research.
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