The following mini-review attempts to guide researchers in the quantification of fluorescently-labelled proteins within cultured thick or chromogenically-stained proteins within thin sections of brain tissue.It follow...The following mini-review attempts to guide researchers in the quantification of fluorescently-labelled proteins within cultured thick or chromogenically-stained proteins within thin sections of brain tissue.It follows from our examination of the utility of Fiji Image J thresholding and binarization algorithms.Describing how we identified the maximum intensity projection as the best of six tested for two dimensional（2 D）-rendering of three-dimensional（3 D） images derived from a series of z-stacked micrographs,the review summarises our comparison of 16 global and 9 local algorithms for their ability to accurately quantify the expression of astrocytic glial fibrillary acidic protein（GFAP）,microglial ionized calcium binding adapter molecule 1（IBA1） and oligodendrocyte lineage Olig2 within fixed cultured rat hippocampal brain slices.The application of these algorithms to chromogenically-stained GFAP and IBA1 within thin tissue sections,is also described.Fiji’s Bio Voxxel plugin allowed categorisation of algorithms according to their sensitivity,specificity accuracy and relative quality.The Percentile algorithm was deemed best for quantifying levels of GFAP,the Li algorithm was best when quantifying IBA expression,while the Otsu algorithm was optimum for Olig2 staining,albeit with over-quantification of oligodendrocyte number when compared to a stereological approach.Also,GFAP and IBA expression in 3,3′-diaminobenzidine（DAB）/haematoxylin-stained cerebellar tissue was best quantified with Default,Isodata and Moments algorithms.The workflow presented in Figure 1 could help to improve the quality of research outcomes that are based on the quantification of protein with brain tissue.展开更多
Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system(CNS)tumors and greatly influence mortality and therapy-induced long-term morbidity in sur...Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system(CNS)tumors and greatly influence mortality and therapy-induced long-term morbidity in survivors.A number of in vitro and ex vivo assay systems have been established to better understand the infiltration and metastatic processes,to search for molecules that specifically block tumor cell infiltration and metastatic dissemination and to pre-clinically evaluate their efficaciousness.These systems allow analytical testing of tumor cell viability and motile and invasive capabilities in simplified and well-controlled environments.However,the urgent need for novel anti-metastatic therapies has provided an incentive for the further development of not only classical in vitro methods but also of novel,physiologically more relevant assay systems including organotypic brain slice culture.In this review,using publicly available peer-reviewed primary research and review articles,we provide an overview of a selection of in vitro and ex vivo techniques widely used to study growth and dissemination of primary metastatic brain tumors.Furthermore,we discuss how our steadily increasing knowledge of tumor biology and the tumor microenvironment could be integrated to improve current research methods for metastatic brain tumors.We believe that such rationally improved methods will ultimately increase our understanding of the biology of brain tumors and facilitate the development of more efficacious anti-metastatic treatments.展开更多
基金supported by a grant from Thomas Crawford Hayes Research Fundthe NUI Galway College of Science scholarship to SHa grant from NUI Galway Foundation Office to JM
文摘The following mini-review attempts to guide researchers in the quantification of fluorescently-labelled proteins within cultured thick or chromogenically-stained proteins within thin sections of brain tissue.It follows from our examination of the utility of Fiji Image J thresholding and binarization algorithms.Describing how we identified the maximum intensity projection as the best of six tested for two dimensional（2 D）-rendering of three-dimensional（3 D） images derived from a series of z-stacked micrographs,the review summarises our comparison of 16 global and 9 local algorithms for their ability to accurately quantify the expression of astrocytic glial fibrillary acidic protein（GFAP）,microglial ionized calcium binding adapter molecule 1（IBA1） and oligodendrocyte lineage Olig2 within fixed cultured rat hippocampal brain slices.The application of these algorithms to chromogenically-stained GFAP and IBA1 within thin tissue sections,is also described.Fiji’s Bio Voxxel plugin allowed categorisation of algorithms according to their sensitivity,specificity accuracy and relative quality.The Percentile algorithm was deemed best for quantifying levels of GFAP,the Li algorithm was best when quantifying IBA expression,while the Otsu algorithm was optimum for Olig2 staining,albeit with over-quantification of oligodendrocyte number when compared to a stereological approach.Also,GFAP and IBA expression in 3,3′-diaminobenzidine（DAB）/haematoxylin-stained cerebellar tissue was best quantified with Default,Isodata and Moments algorithms.The workflow presented in Figure 1 could help to improve the quality of research outcomes that are based on the quantification of protein with brain tissue.
文摘Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system(CNS)tumors and greatly influence mortality and therapy-induced long-term morbidity in survivors.A number of in vitro and ex vivo assay systems have been established to better understand the infiltration and metastatic processes,to search for molecules that specifically block tumor cell infiltration and metastatic dissemination and to pre-clinically evaluate their efficaciousness.These systems allow analytical testing of tumor cell viability and motile and invasive capabilities in simplified and well-controlled environments.However,the urgent need for novel anti-metastatic therapies has provided an incentive for the further development of not only classical in vitro methods but also of novel,physiologically more relevant assay systems including organotypic brain slice culture.In this review,using publicly available peer-reviewed primary research and review articles,we provide an overview of a selection of in vitro and ex vivo techniques widely used to study growth and dissemination of primary metastatic brain tumors.Furthermore,we discuss how our steadily increasing knowledge of tumor biology and the tumor microenvironment could be integrated to improve current research methods for metastatic brain tumors.We believe that such rationally improved methods will ultimately increase our understanding of the biology of brain tumors and facilitate the development of more efficacious anti-metastatic treatments.
