RESUMO
Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.
Assuntos
Anticorpos , Proteômica , Proteômica/métodos , Reprodutibilidade dos Testes , Imunofluorescência , BiomarcadoresRESUMO
OBJECTIVE: Alzheimer disease (AD) is the most common form of neurodegenerative disease in elderly people. Toxic brain amyloid-beta (Aß) aggregates and ensuing cell death are believed to play a central role in the pathogenesis of the disease. In this study, we investigated if we could monitor the presence of these aggregates by performing in situ electrical impedance spectroscopy measurements in AD model mice brains. APPROACH: In this study, electrical impedance spectroscopy measurements were performed post-mortem in APPPS1 transgenic mice brains. This transgenic model is commonly used to study amyloidogenesis, a pathological hallmark of AD. We used flexible probes with embedded micrometric electrodes array to demonstrate the feasibility of detecting senile plaques composed of Aß peptides by localized impedance measurements. MAIN RESULTS: We particularly focused on deep brain structures, such as the hippocampus. Ex vivo experiments using brains from young and old APPPS1 mice lead us to show that impedance measurements clearly correlate with the percentage of Aß plaque load in the brain tissues. We could monitor the effects of aging in the AD APPPS1 mice model. SIGNIFICANCE: We demonstrated that a localized electrical impedance measurement constitutes a valuable technique to monitor the presence of Aß-plaques, which is complementary with existing imaging techniques. This method does not require prior Aß staining, precluding the risk of variations in tissue uptake of dyes or tracers, and consequently ensuring reproducible data collection.
Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Espectroscopia Dielétrica/métodos , Placa Amiloide/metabolismo , Animais , Técnicas de Diagnóstico Neurológico/instrumentação , Espectroscopia Dielétrica/instrumentação , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Distribuição TecidualRESUMO
Electrical impedance spectroscopy measurements were performed in post-mortem mice brains using a flexible probe with an embedded micrometric electrode array. Combined with a peak resistance frequency method this allowed obtaining intrinsic resistivity values of brain tissues and structures with submillimetric resolution. Reproducible resistivity measurements are reported, which allows the resistivity in the cortex, ventricle, fiber tracts, thalamus and basal ganglia to be differentiated. Measurements of brain slices revealed resistivity profiles correlated with the local density of cell bodies hence allowing to discriminate between the different cortical layers. Finally, impedance measurements were performed on a model of cauterized mouse brain evidencing the possibility to measure the spatial extent and the degree of the tissue denaturation due to the cauterization.
Assuntos
Mapeamento Encefálico/instrumentação , Encéfalo/citologia , Encéfalo/fisiologia , Condutometria/instrumentação , Análise em Microsséries/instrumentação , Microeletrodos , Pletismografia de Impedância/instrumentação , Animais , Contagem de Células/instrumentação , Impedância Elétrica , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We present a novel technology for the simultaneous and simple impedimetric screening of multiple microfluidic channels with only one electrode pair. We have exploited the frequency dimension to distinguish between up to three channels. Each 'sub-sensor' possesses its corresponding measurement frequency where the sample-specific dielectric properties can be probed. We have shown the validity of our frequency-multiplexing impedance sensor (FMIS) by comparison with conventional 'single sensors'. Our highly sensitive FMIS was proven suitable for life science applications through usage as a cell-based toxicology platform. We are confident that our technology might find great utility in parallelized cell-based analysis systems as well as in biomedical devices where size limitations and spatially distributed probing are important parameters.