PMC:5118445 / 892-4802 JSONTXT

{"project":"TEST0","denotations":[{"id":"27920776-89-94-3175554","span":{"begin":102,"end":103},"obj":"[\"15001782\"]"},{"id":"27920776-238-243-3175555","span":{"begin":349,"end":350},"obj":"[\"27135878\"]"},{"id":"27920776-77-82-3175556","span":{"begin":430,"end":431},"obj":"[\"19876394\"]"},{"id":"27920776-210-215-3175557","span":{"begin":644,"end":645},"obj":"[\"17210947\"]"},{"id":"27920776-213-218-3175558","span":{"begin":647,"end":648},"obj":"[\"20974816\"]"},{"id":"27920776-49-54-3175559","span":{"begin":700,"end":701},"obj":"[\"18209087\"]"},{"id":"27920776-52-57-3175560","span":{"begin":703,"end":704},"obj":"[\"19153223\"]"},{"id":"27920776-72-77-3175561","span":{"begin":1007,"end":1008},"obj":"[\"23316198\", \"23824885\", \"24504961\", \"25792554\", \"26925759\"]"},{"id":"27920776-149-155-3175562","span":{"begin":1306,"end":1308},"obj":"[\"22945932\"]"},{"id":"27920776-166-172-3175563","span":{"begin":1622,"end":1624},"obj":"[\"26205971\"]"},{"id":"27920776-170-176-3175564","span":{"begin":1626,"end":1628},"obj":"[\"18312656\"]"},{"id":"27920776-124-130-3175565","span":{"begin":2491,"end":2493},"obj":"[\"14767483\"]"},{"id":"27920776-128-134-3175566","span":{"begin":2495,"end":2497},"obj":"[\"15637334\"]"},{"id":"27920776-192-198-3175567","span":{"begin":2692,"end":2694},"obj":"[\"7506771\"]"},{"id":"27920776-196-202-3175568","span":{"begin":2696,"end":2698},"obj":"[\"7822775\"]"},{"id":"27920776-226-232-3175569","span":{"begin":3035,"end":3037},"obj":"[\"19886757\"]"}],"text":"Introduction\nNeutrophil extracellular traps (NETs) probably evolved to counteract invading pathogens (1). When their production or degradation is not controlled, they have pathogenic potential and are involved in numerous diseases including autoimmunity, thrombosis, lung diseases, infertility, diabetes, cancer, and neurodegeneration, reviewed in (2). NETs are composed of chromatin, granular, and some cytoplasmic constituents (3). In naive neutrophils, granular and nuclear antigens are spatially separated and during NETosis, some granular proteins, like neutrophil elastase (NE) and myeloperoxidase (MPO) gradually migrate to the nucleus (4, 5). Also, histones are citrullinated during NETosis (6, 7). Thus, the localization of granular proteins in the nucleus and the citrullination of histones provide unique feature for neutrophils undergoing NETosis that can be exploited for identification of these cells in tissue sections.\nIn vitro, there are a variety of protocols to detect and quantify NETs (8–12). It is important to note that the widely spread out strands associated with NET images generated in vitro are probably an artifact of fixation. Indeed, in life cell imaging studies with isolated neutrophils, NETs appear as a diffuse cloud formed by the strands of NETs floating in the medium (13). It is not clear how NETs appear in tissues, where space is restricted, and the NETs are unlikely to appear as the large areas observed in vitro. Classical histological stains, like hematoxylin/eosin, may indicate the presence of NETs, but there are only few examples of NETs detection with histological stains (14, 15). Notably, there is no general protocol to identify NETs in tissues.\nWhile they are well suited for staining with most antibodies, cryo sections from freshly frozen tissue have the disadvantage that due to ice crystal formation neutrophils in the tissue can be damaged, thus NET-like structures can be generated as a preparation artifact. In contrast, fixation with buffered paraformaldehyde solution, ideally by perfusion, preserves the tissue architecture including NETs. Here, we present methods for formalin-fixed and paraffin-embedded sections. These sections are available from pathological studies and can be conserved indefinitely before analysis.\nMost antibodies will not readily bind to their epitopes in formalin-fixed tissue. The reason for this is the formation of intra- and intermolecular cross-links by methylene bridges that mask most epitopes (16, 17). For successful immunohistological staining, antigen retrieval is required that normally involves heating of the rehydrated sections in a suitable heat-induced epitope retrieval (HIER) buffer (18, 19). This breaks the methylene bridges that prevent binding of the antibody and renders the epitopes accessible. In a study with histopathologically important antibodies, it was shown that most epitopes detected by clinically relevant antibodies are linear and can be reversibly blocked by binding to neighboring proteins during fixation (20).\nWe tested a series of antibodies against NET components for their ability to bind to their epitopes in formalin-fixed paraffin-embedded tissue. We selected nine antibodies with good staining properties and tested various antigen retrieval methods to find suitable combinations for double or triple immunofluorescence. In this paper, we present protocols that allow simultaneous staining for nuclear and granular or cytoplasmic NET components in paraffin-embedded tissue sections after antigen retrieval.\nFor the identification of NETs it is necessary to determine if nuclear antigens are colocalized with granular and/or cytoplasmic components. Hence, micrographs have to be prepared at a primary magnification of at least 20×. The resulting images can be used to quantify the fluorescent signals as an unbiased means for the detection and measurement of NETs in tissue."}

{"project":"2_test","denotations":[{"id":"27920776-15001782-34775593","span":{"begin":102,"end":103},"obj":"15001782"},{"id":"27920776-27135878-34775594","span":{"begin":349,"end":350},"obj":"27135878"},{"id":"27920776-19876394-34775595","span":{"begin":430,"end":431},"obj":"19876394"},{"id":"27920776-17210947-34775596","span":{"begin":644,"end":645},"obj":"17210947"},{"id":"27920776-20974816-34775597","span":{"begin":647,"end":648},"obj":"20974816"},{"id":"27920776-18209087-34775598","span":{"begin":700,"end":701},"obj":"18209087"},{"id":"27920776-19153223-34775599","span":{"begin":703,"end":704},"obj":"19153223"},{"id":"27920776-23316198-34775600","span":{"begin":1007,"end":1008},"obj":"23316198"},{"id":"27920776-23824885-34775600","span":{"begin":1007,"end":1008},"obj":"23824885"},{"id":"27920776-24504961-34775600","span":{"begin":1007,"end":1008},"obj":"24504961"},{"id":"27920776-25792554-34775600","span":{"begin":1007,"end":1008},"obj":"25792554"},{"id":"27920776-26925759-34775600","span":{"begin":1007,"end":1008},"obj":"26925759"},{"id":"27920776-22945932-34775601","span":{"begin":1306,"end":1308},"obj":"22945932"},{"id":"27920776-26205971-34775602","span":{"begin":1622,"end":1624},"obj":"26205971"},{"id":"27920776-18312656-34775603","span":{"begin":1626,"end":1628},"obj":"18312656"},{"id":"27920776-14767483-34775604","span":{"begin":2491,"end":2493},"obj":"14767483"},{"id":"27920776-15637334-34775605","span":{"begin":2495,"end":2497},"obj":"15637334"},{"id":"27920776-7506771-34775606","span":{"begin":2692,"end":2694},"obj":"7506771"},{"id":"27920776-7822775-34775607","span":{"begin":2696,"end":2698},"obj":"7822775"},{"id":"27920776-19886757-34775608","span":{"begin":3035,"end":3037},"obj":"19886757"}],"text":"Introduction\nNeutrophil extracellular traps (NETs) probably evolved to counteract invading pathogens (1). When their production or degradation is not controlled, they have pathogenic potential and are involved in numerous diseases including autoimmunity, thrombosis, lung diseases, infertility, diabetes, cancer, and neurodegeneration, reviewed in (2). NETs are composed of chromatin, granular, and some cytoplasmic constituents (3). In naive neutrophils, granular and nuclear antigens are spatially separated and during NETosis, some granular proteins, like neutrophil elastase (NE) and myeloperoxidase (MPO) gradually migrate to the nucleus (4, 5). Also, histones are citrullinated during NETosis (6, 7). Thus, the localization of granular proteins in the nucleus and the citrullination of histones provide unique feature for neutrophils undergoing NETosis that can be exploited for identification of these cells in tissue sections.\nIn vitro, there are a variety of protocols to detect and quantify NETs (8–12). It is important to note that the widely spread out strands associated with NET images generated in vitro are probably an artifact of fixation. Indeed, in life cell imaging studies with isolated neutrophils, NETs appear as a diffuse cloud formed by the strands of NETs floating in the medium (13). It is not clear how NETs appear in tissues, where space is restricted, and the NETs are unlikely to appear as the large areas observed in vitro. Classical histological stains, like hematoxylin/eosin, may indicate the presence of NETs, but there are only few examples of NETs detection with histological stains (14, 15). Notably, there is no general protocol to identify NETs in tissues.\nWhile they are well suited for staining with most antibodies, cryo sections from freshly frozen tissue have the disadvantage that due to ice crystal formation neutrophils in the tissue can be damaged, thus NET-like structures can be generated as a preparation artifact. In contrast, fixation with buffered paraformaldehyde solution, ideally by perfusion, preserves the tissue architecture including NETs. Here, we present methods for formalin-fixed and paraffin-embedded sections. These sections are available from pathological studies and can be conserved indefinitely before analysis.\nMost antibodies will not readily bind to their epitopes in formalin-fixed tissue. The reason for this is the formation of intra- and intermolecular cross-links by methylene bridges that mask most epitopes (16, 17). For successful immunohistological staining, antigen retrieval is required that normally involves heating of the rehydrated sections in a suitable heat-induced epitope retrieval (HIER) buffer (18, 19). This breaks the methylene bridges that prevent binding of the antibody and renders the epitopes accessible. In a study with histopathologically important antibodies, it was shown that most epitopes detected by clinically relevant antibodies are linear and can be reversibly blocked by binding to neighboring proteins during fixation (20).\nWe tested a series of antibodies against NET components for their ability to bind to their epitopes in formalin-fixed paraffin-embedded tissue. We selected nine antibodies with good staining properties and tested various antigen retrieval methods to find suitable combinations for double or triple immunofluorescence. In this paper, we present protocols that allow simultaneous staining for nuclear and granular or cytoplasmic NET components in paraffin-embedded tissue sections after antigen retrieval.\nFor the identification of NETs it is necessary to determine if nuclear antigens are colocalized with granular and/or cytoplasmic components. Hence, micrographs have to be prepared at a primary magnification of at least 20×. The resulting images can be used to quantify the fluorescent signals as an unbiased means for the detection and measurement of NETs in tissue."}

{"project":"MyTest","denotations":[{"id":"27920776-15001782-34775593","span":{"begin":102,"end":103},"obj":"15001782"},{"id":"27920776-27135878-34775594","span":{"begin":349,"end":350},"obj":"27135878"},{"id":"27920776-19876394-34775595","span":{"begin":430,"end":431},"obj":"19876394"},{"id":"27920776-17210947-34775596","span":{"begin":644,"end":645},"obj":"17210947"},{"id":"27920776-20974816-34775597","span":{"begin":647,"end":648},"obj":"20974816"},{"id":"27920776-18209087-34775598","span":{"begin":700,"end":701},"obj":"18209087"},{"id":"27920776-19153223-34775599","span":{"begin":703,"end":704},"obj":"19153223"},{"id":"27920776-23316198-34775600","span":{"begin":1007,"end":1008},"obj":"23316198"},{"id":"27920776-23824885-34775600","span":{"begin":1007,"end":1008},"obj":"23824885"},{"id":"27920776-24504961-34775600","span":{"begin":1007,"end":1008},"obj":"24504961"},{"id":"27920776-25792554-34775600","span":{"begin":1007,"end":1008},"obj":"25792554"},{"id":"27920776-26925759-34775600","span":{"begin":1007,"end":1008},"obj":"26925759"},{"id":"27920776-22945932-34775601","span":{"begin":1306,"end":1308},"obj":"22945932"},{"id":"27920776-26205971-34775602","span":{"begin":1622,"end":1624},"obj":"26205971"},{"id":"27920776-18312656-34775603","span":{"begin":1626,"end":1628},"obj":"18312656"},{"id":"27920776-14767483-34775604","span":{"begin":2491,"end":2493},"obj":"14767483"},{"id":"27920776-15637334-34775605","span":{"begin":2495,"end":2497},"obj":"15637334"},{"id":"27920776-7506771-34775606","span":{"begin":2692,"end":2694},"obj":"7506771"},{"id":"27920776-7822775-34775607","span":{"begin":2696,"end":2698},"obj":"7822775"},{"id":"27920776-19886757-34775608","span":{"begin":3035,"end":3037},"obj":"19886757"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"Introduction\nNeutrophil extracellular traps (NETs) probably evolved to counteract invading pathogens (1). When their production or degradation is not controlled, they have pathogenic potential and are involved in numerous diseases including autoimmunity, thrombosis, lung diseases, infertility, diabetes, cancer, and neurodegeneration, reviewed in (2). NETs are composed of chromatin, granular, and some cytoplasmic constituents (3). In naive neutrophils, granular and nuclear antigens are spatially separated and during NETosis, some granular proteins, like neutrophil elastase (NE) and myeloperoxidase (MPO) gradually migrate to the nucleus (4, 5). Also, histones are citrullinated during NETosis (6, 7). Thus, the localization of granular proteins in the nucleus and the citrullination of histones provide unique feature for neutrophils undergoing NETosis that can be exploited for identification of these cells in tissue sections.\nIn vitro, there are a variety of protocols to detect and quantify NETs (8–12). It is important to note that the widely spread out strands associated with NET images generated in vitro are probably an artifact of fixation. Indeed, in life cell imaging studies with isolated neutrophils, NETs appear as a diffuse cloud formed by the strands of NETs floating in the medium (13). It is not clear how NETs appear in tissues, where space is restricted, and the NETs are unlikely to appear as the large areas observed in vitro. Classical histological stains, like hematoxylin/eosin, may indicate the presence of NETs, but there are only few examples of NETs detection with histological stains (14, 15). Notably, there is no general protocol to identify NETs in tissues.\nWhile they are well suited for staining with most antibodies, cryo sections from freshly frozen tissue have the disadvantage that due to ice crystal formation neutrophils in the tissue can be damaged, thus NET-like structures can be generated as a preparation artifact. In contrast, fixation with buffered paraformaldehyde solution, ideally by perfusion, preserves the tissue architecture including NETs. Here, we present methods for formalin-fixed and paraffin-embedded sections. These sections are available from pathological studies and can be conserved indefinitely before analysis.\nMost antibodies will not readily bind to their epitopes in formalin-fixed tissue. The reason for this is the formation of intra- and intermolecular cross-links by methylene bridges that mask most epitopes (16, 17). For successful immunohistological staining, antigen retrieval is required that normally involves heating of the rehydrated sections in a suitable heat-induced epitope retrieval (HIER) buffer (18, 19). This breaks the methylene bridges that prevent binding of the antibody and renders the epitopes accessible. In a study with histopathologically important antibodies, it was shown that most epitopes detected by clinically relevant antibodies are linear and can be reversibly blocked by binding to neighboring proteins during fixation (20).\nWe tested a series of antibodies against NET components for their ability to bind to their epitopes in formalin-fixed paraffin-embedded tissue. We selected nine antibodies with good staining properties and tested various antigen retrieval methods to find suitable combinations for double or triple immunofluorescence. In this paper, we present protocols that allow simultaneous staining for nuclear and granular or cytoplasmic NET components in paraffin-embedded tissue sections after antigen retrieval.\nFor the identification of NETs it is necessary to determine if nuclear antigens are colocalized with granular and/or cytoplasmic components. Hence, micrographs have to be prepared at a primary magnification of at least 20×. The resulting images can be used to quantify the fluorescent signals as an unbiased means for the detection and measurement of NETs in tissue."}