RESUMO
In the last decades, the variety of programs, algorithms, and strategies that researchers have at their disposal to process and analyze image files has grown extensively. However, these are only pointless tools if not applied with the careful planning required to achieve a succesful image analysis. In order to do so, the analyst must establish a meaningful and effective sequence of orderly operations that is able to (1) overcome all the problems derived from the image manipulation and (2) successfully resolve the question that was originally posed. In this chapter, the authors suggest a set of strategies and present a reflection on the main milestones that compose the image processing workflow, to help guide the way to obtaining unbiased quantitative data.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Fluxo de Trabalho , Algoritmos , Animais , Microscopia/instrumentação , SoftwareRESUMO
Pulse-chase experiments using 5-bromo-2'-deoxyuridine (BrdU), or the more recent EdU (5-etynil-2'-deoxyuridine), enable the identification of cells going through S phase. This chapter describes a high-content proliferation assay pipeline for adherent cell cultures. High-throughput imaging is followed by high-content data analysis using a non-supervised ImageJ macroinstruction that segments the individual nuclei, determines the nucleoside analogue absence/presence, and measures the signal of up to two additional nuclear markers. Based upon the specific combination with proliferation-specific protein immunostaining, the percentage of cells undergoing different phases of the cell cycle (G0, G1, S, G2, and M) might be established. The method can be also used to estimate the proliferation (S phase) rate of particular cell subpopulations identified through labelling with specific nuclear markers.
Assuntos
Proliferação de Células/fisiologia , Corantes Fluorescentes/química , Processamento de Imagem Assistida por Computador/métodos , Software , Bromodesoxiuridina/química , Técnicas de Cultura de Células , Ciclo Celular , Núcleo Celular/química , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Conjuntos de Dados como Assunto , Indóis/químicaRESUMO
Autosomal dominant lateral temporal epilepsy (EPT; OMIM 600512) is a form of epilepsy characterized by partial seizures, usually preceded by auditory signs. The gene for this disorder has been mapped by linkage studies to chromosomal region 10q24. Here we show that mutations in the LGI1 gene segregate with EPT in two families affected by this disorder. Both mutations introduce premature stop codons and thus prevent the production of the full-length protein from the affected allele. By immunohistochemical studies, we demonstrate that the LGI1 protein, which contains several leucine-rich repeats, is expressed ubiquitously in the neuronal cell compartment of the brain. Moreover, we provide evidence for genetic heterogeneity within this disorder, since several other families with a phenotype consistent with this type of epilepsy lack mutations in the LGI1 gene.