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cell preparation and immunostanining\nHippocampal neuron cultures were prepared as previously reported [6]. In brief, the hippocampuses were dissected out from mouse embryos at 16 days in utero. The tissues were then incubated for 20 min at 37°C in MEM (minimum essential medium) modified for suspension culture (Life Technologies) plus 0.25% trypsin (Life Technologies). The dissociated hippocampal neurons were plated on glass coverslips coated with a confluent monolayer of mouse cortical astrocytes obtained as described below. The neurons were maintained at 37°C in a humidified atmosphere with 5% CO2. Cortical astrocytes dissociated from newborn mouse cortices were grown in culture flasks at 37°C in a humidified atmosphere with 5% CO2 until confluent. The cells were exposed to 10-5 M cytosine arabinoside (Sigma) and cultured for additional 12–24 hrs at 37°C. After remove of the media with cellular debris, the cells were used for coating coverslips.\nFor immunostaining, neuronal cells on the coverslips were first fixed in PBS containing 4% paraformaldehyde (PFA) for 12 hrs at 4°C and then incubated in a solution containing 4% PFA and 0.4% Triton X-100 at 4°C for 1 hr. After washing with PBS three times, the cells were incubated with a blocking solution containing 1:30 normal goat serum, and subsequently incubated with a rabbit polyclonal anti-ACDP antibody (1:3000) overnight at 4°C. After extensive washing with 1% goat serum PBS solution, the cells were incubated with an Alex 488 conjugated secondary antibody (1:100 in 1% goat serum PBS solution, Molecular Probes) for 3 hrs at room temperature. Following final washes with 1% goat serum PBS solution, the neuronal cells on the coverslips were cover-slipped with a glycerol-based anti-photobleach medium. The cells were viewed under a confocal microscope (Carl Zeiss). Images were captured with a CCD camera and acquired by the Scion Image software (Scion Corporation, Frederick, MD)."}

craft-ca-core-dev

{"project":"craft-ca-core-dev","denotations":[{"id":"T5207","span":{"begin":1746,"end":1754},"obj":"CHEBI:17754"},{"id":"T5206","span":{"begin":1687,"end":1701},"obj":"CL:0000540"},{"id":"T5205","span":{"begin":1673,"end":1681},"obj":"CHEBI:75958"},{"id":"T5204","span":{"begin":1658,"end":1662},"obj":"NCBITaxon:9925"},{"id":"T5203","span":{"begin":1568,"end":1576},"obj":"CHEBI:75958"},{"id":"T5202","span":{"begin":1553,"end":1557},"obj":"NCBITaxon:9925"},{"id":"T5201","span":{"begin":1531,"end":1539},"obj":"GO:0042571"},{"id":"T5200","span":{"begin":1510,"end":1520},"obj":"MOP:0000779"},{"id":"T5199","span":{"begin":1501,"end":1509},"obj":"CHEBI:52661"},{"id":"T5198","span":{"begin":1458,"end":1466},"obj":"CHEBI:75958"},{"id":"T5197","span":{"begin":1443,"end":1447},"obj":"NCBITaxon:9925"},{"id":"T5196","span":{"begin":1375,"end":1383},"obj":"GO:0042571"},{"id":"T5195","span":{"begin":1347,"end":1353},"obj":"NCBITaxon:9986"},{"id":"T5194","span":{"begin":1301,"end":1305},"obj":"NCBITaxon:9925"},{"id":"T5193","span":{"begin":1269,"end":1277},"obj":"CHEBI:75958"},{"id":"T5192","span":{"begin":1162,"end":1174},"obj":"CHEBI:9750"},{"id":"T5191","span":{"begin":1126,"end":1134},"obj":"CHEBI:75958"},{"id":"T5190","span":{"begin":990,"end":1004},"obj":"CL:0000540"},{"id":"T5189","span":{"begin":802,"end":822},"obj":"CHEBI:28680"},{"id":"T5188","span":{"begin":748,"end":751},"obj":"CHEBI:16526"},{"id":"T5187","span":{"begin":681,"end":686},"obj":"GO:0040007"},{"id":"T5186","span":{"begin":667,"end":675},"obj":"UBERON:0001851"},{"id":"T5185","span":{"begin":661,"end":666},"obj":"NCBITaxon:10088"},{"id":"T5184","span":{"begin":616,"end":635},"obj":"CL:0002605"},{"id":"T5183","span":{"begin":616,"end":624},"obj":"UBERON:0001851"},{"id":"T5182","span":{"begin":611,"end":614},"obj":"CHEBI:16526"},{"id":"T5181","span":{"begin":544,"end":551},"obj":"CL:0000540"},{"id":"T5180","span":{"begin":491,"end":510},"obj":"CL:0002605"},{"id":"T5179","span":{"begin":491,"end":499},"obj":"UBERON:0001851"},{"id":"T5178","span":{"begin":485,"end":490},"obj":"NCBITaxon:10088"},{"id":"T5177","span":{"begin":396,"end":415},"obj":"CL:0002608"},{"id":"T5176","span":{"begin":351,"end":358},"obj":"PR:000027795"},{"id":"T5175","span":{"begin":207,"end":214},"obj":"UBERON:0000479"},{"id":"T5174","span":{"begin":174,"end":181},"obj":"UBERON:0000922"},{"id":"T5173","span":{"begin":168,"end":173},"obj":"NCBITaxon:10088"},{"id":"T5172","span":{"begin":46,"end":64},"obj":"CL:0002608"},{"id":"T5171","span":{"begin":0,"end":13},"obj":"CL:0000540"}],"text":"Neuronal cell preparation and immunostanining\nHippocampal neuron cultures were prepared as previously reported [6]. In brief, the hippocampuses were dissected out from mouse embryos at 16 days in utero. The tissues were then incubated for 20 min at 37°C in MEM (minimum essential medium) modified for suspension culture (Life Technologies) plus 0.25% trypsin (Life Technologies). The dissociated hippocampal neurons were plated on glass coverslips coated with a confluent monolayer of mouse cortical astrocytes obtained as described below. The neurons were maintained at 37°C in a humidified atmosphere with 5% CO2. Cortical astrocytes dissociated from newborn mouse cortices were grown in culture flasks at 37°C in a humidified atmosphere with 5% CO2 until confluent. The cells were exposed to 10-5 M cytosine arabinoside (Sigma) and cultured for additional 12–24 hrs at 37°C. After remove of the media with cellular debris, the cells were used for coating coverslips.\nFor immunostaining, neuronal cells on the coverslips were first fixed in PBS containing 4% paraformaldehyde (PFA) for 12 hrs at 4°C and then incubated in a solution containing 4% PFA and 0.4% Triton X-100 at 4°C for 1 hr. After washing with PBS three times, the cells were incubated with a blocking solution containing 1:30 normal goat serum, and subsequently incubated with a rabbit polyclonal anti-ACDP antibody (1:3000) overnight at 4°C. After extensive washing with 1% goat serum PBS solution, the cells were incubated with an Alex 488 conjugated secondary antibody (1:100 in 1% goat serum PBS solution, Molecular Probes) for 3 hrs at room temperature. Following final washes with 1% goat serum PBS solution, the neuronal cells on the coverslips were cover-slipped with a glycerol-based anti-photobleach medium. The cells were viewed under a confocal microscope (Carl Zeiss). Images were captured with a CCD camera and acquired by the Scion Image software (Scion Corporation, Frederick, MD)."}

2_test

{"project":"2_test","denotations":[{"id":"14723793-8657283-10642200","span":{"begin":112,"end":113},"obj":"8657283"}],"text":"Neuronal cell preparation and immunostanining\nHippocampal neuron cultures were prepared as previously reported [6]. In brief, the hippocampuses were dissected out from mouse embryos at 16 days in utero. The tissues were then incubated for 20 min at 37°C in MEM (minimum essential medium) modified for suspension culture (Life Technologies) plus 0.25% trypsin (Life Technologies). The dissociated hippocampal neurons were plated on glass coverslips coated with a confluent monolayer of mouse cortical astrocytes obtained as described below. The neurons were maintained at 37°C in a humidified atmosphere with 5% CO2. Cortical astrocytes dissociated from newborn mouse cortices were grown in culture flasks at 37°C in a humidified atmosphere with 5% CO2 until confluent. The cells were exposed to 10-5 M cytosine arabinoside (Sigma) and cultured for additional 12–24 hrs at 37°C. After remove of the media with cellular debris, the cells were used for coating coverslips.\nFor immunostaining, neuronal cells on the coverslips were first fixed in PBS containing 4% paraformaldehyde (PFA) for 12 hrs at 4°C and then incubated in a solution containing 4% PFA and 0.4% Triton X-100 at 4°C for 1 hr. After washing with PBS three times, the cells were incubated with a blocking solution containing 1:30 normal goat serum, and subsequently incubated with a rabbit polyclonal anti-ACDP antibody (1:3000) overnight at 4°C. After extensive washing with 1% goat serum PBS solution, the cells were incubated with an Alex 488 conjugated secondary antibody (1:100 in 1% goat serum PBS solution, Molecular Probes) for 3 hrs at room temperature. Following final washes with 1% goat serum PBS solution, the neuronal cells on the coverslips were cover-slipped with a glycerol-based anti-photobleach medium. The cells were viewed under a confocal microscope (Carl Zeiss). Images were captured with a CCD camera and acquired by the Scion Image software (Scion Corporation, Frederick, MD)."}

craft-ca-core-ex-dev

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T5263","span":{"begin":1790,"end":1795},"obj":"CL_GO_EXT:cell"},{"id":"T5262","span":{"begin":1746,"end":1754},"obj":"CHEBI:17754"},{"id":"T5261","span":{"begin":1696,"end":1701},"obj":"CL_GO_EXT:cell"},{"id":"T5260","span":{"begin":1687,"end":1701},"obj":"CL:0000540"},{"id":"T5259","span":{"begin":1673,"end":1681},"obj":"CHEBI:75958"},{"id":"T5258","span":{"begin":1658,"end":1662},"obj":"NCBITaxon:9925"},{"id":"T5257","span":{"begin":1588,"end":1594},"obj":"CHEBI_SO_EXT:molecular_probe"},{"id":"T5256","span":{"begin":1578,"end":1587},"obj":"CHEBI_EXT:polyatomic_entity_or_group"},{"id":"T5255","span":{"begin":1568,"end":1576},"obj":"CHEBI:75958"},{"id":"T5254","span":{"begin":1553,"end":1557},"obj":"NCBITaxon:9925"},{"id":"T5253","span":{"begin":1531,"end":1539},"obj":"GO:0042571"},{"id":"T5252","span":{"begin":1510,"end":1520},"obj":"MOP:0000779"},{"id":"T5251","span":{"begin":1501,"end":1509},"obj":"CHEBI:52661"},{"id":"T5250","span":{"begin":1472,"end":1477},"obj":"CL_GO_EXT:cell"},{"id":"T5249","span":{"begin":1458,"end":1466},"obj":"CHEBI:75958"},{"id":"T5248","span":{"begin":1443,"end":1447},"obj":"NCBITaxon:9925"},{"id":"T5247","span":{"begin":1375,"end":1383},"obj":"GO:0042571"},{"id":"T5246","span":{"begin":1370,"end":1374},"obj":"PR_EXT:ACDP_or_CNNM"},{"id":"T5245","span":{"begin":1347,"end":1353},"obj":"NCBITaxon:9986"},{"id":"T5244","span":{"begin":1301,"end":1305},"obj":"NCBITaxon:9925"},{"id":"T5243","span":{"begin":1269,"end":1277},"obj":"CHEBI:75958"},{"id":"T5242","span":{"begin":1232,"end":1237},"obj":"CL_GO_EXT:cell"},{"id":"T5241","span":{"begin":1162,"end":1174},"obj":"CHEBI:9750"},{"id":"T5240","span":{"begin":1149,"end":1152},"obj":"CHEBI_EXT:paraformaldehye"},{"id":"T5239","span":{"begin":1126,"end":1134},"obj":"CHEBI:75958"},{"id":"T5238","span":{"begin":1079,"end":1082},"obj":"CHEBI_EXT:paraformaldehye"},{"id":"T5237","span":{"begin":1061,"end":1077},"obj":"CHEBI_EXT:paraformaldehye"},{"id":"T5236","span":{"begin":999,"end":1004},"obj":"CL_GO_EXT:cell"},{"id":"T5235","span":{"begin":990,"end":1004},"obj":"CL:0000540"},{"id":"T5234","span":{"begin":930,"end":935},"obj":"CL_GO_EXT:cell"},{"id":"T5233","span":{"begin":909,"end":917},"obj":"CL_GO_EXT:cell"},{"id":"T5232","span":{"begin":802,"end":822},"obj":"CHEBI:28680"},{"id":"T5231","span":{"begin":773,"end":778},"obj":"CL_GO_EXT:cell"},{"id":"T5230","span":{"begin":748,"end":751},"obj":"CHEBI:16526"},{"id":"T5229","span":{"begin":681,"end":686},"obj":"GO:0040007"},{"id":"T5228","span":{"begin":667,"end":675},"obj":"UBERON:0001851"},{"id":"T5227","span":{"begin":661,"end":666},"obj":"NCBITaxon:10088"},{"id":"T5226","span":{"begin":653,"end":660},"obj":"PATO_UBERON_EXT:neonate_or_newborn"},{"id":"T5225","span":{"begin":616,"end":635},"obj":"CL:0002605"},{"id":"T5224","span":{"begin":616,"end":624},"obj":"UBERON:0001851"},{"id":"T5223","span":{"begin":611,"end":614},"obj":"CHEBI:16526"},{"id":"T5222","span":{"begin":544,"end":551},"obj":"CL:0000540"},{"id":"T5221","span":{"begin":491,"end":510},"obj":"CL:0002605"},{"id":"T5220","span":{"begin":491,"end":499},"obj":"UBERON:0001851"},{"id":"T5219","span":{"begin":485,"end":490},"obj":"NCBITaxon:10088"},{"id":"T5218","span":{"begin":396,"end":415},"obj":"CL:0002608"},{"id":"T5217","span":{"begin":396,"end":407},"obj":"UBERON_EXT:hippocampus_proper_or_hippocampal_formation"},{"id":"T5216","span":{"begin":351,"end":358},"obj":"PR_EXT:000027795"},{"id":"T5215","span":{"begin":207,"end":214},"obj":"UBERON:0000479"},{"id":"T5214","span":{"begin":174,"end":181},"obj":"UBERON:0000922"},{"id":"T5213","span":{"begin":168,"end":173},"obj":"NCBITaxon:10088"},{"id":"T5212","span":{"begin":130,"end":143},"obj":"UBERON_EXT:hippocampus_proper_or_hippocampal_formation"},{"id":"T5211","span":{"begin":46,"end":64},"obj":"CL:0002608"},{"id":"T5210","span":{"begin":46,"end":57},"obj":"UBERON_EXT:hippocampus_proper_or_hippocampal_formation"},{"id":"T5209","span":{"begin":9,"end":13},"obj":"CL_GO_EXT:cell"},{"id":"T5208","span":{"begin":0,"end":13},"obj":"CL:0000540"}],"text":"Neuronal cell preparation and immunostanining\nHippocampal neuron cultures were prepared as previously reported [6]. In brief, the hippocampuses were dissected out from mouse embryos at 16 days in utero. The tissues were then incubated for 20 min at 37°C in MEM (minimum essential medium) modified for suspension culture (Life Technologies) plus 0.25% trypsin (Life Technologies). The dissociated hippocampal neurons were plated on glass coverslips coated with a confluent monolayer of mouse cortical astrocytes obtained as described below. The neurons were maintained at 37°C in a humidified atmosphere with 5% CO2. Cortical astrocytes dissociated from newborn mouse cortices were grown in culture flasks at 37°C in a humidified atmosphere with 5% CO2 until confluent. The cells were exposed to 10-5 M cytosine arabinoside (Sigma) and cultured for additional 12–24 hrs at 37°C. After remove of the media with cellular debris, the cells were used for coating coverslips.\nFor immunostaining, neuronal cells on the coverslips were first fixed in PBS containing 4% paraformaldehyde (PFA) for 12 hrs at 4°C and then incubated in a solution containing 4% PFA and 0.4% Triton X-100 at 4°C for 1 hr. After washing with PBS three times, the cells were incubated with a blocking solution containing 1:30 normal goat serum, and subsequently incubated with a rabbit polyclonal anti-ACDP antibody (1:3000) overnight at 4°C. After extensive washing with 1% goat serum PBS solution, the cells were incubated with an Alex 488 conjugated secondary antibody (1:100 in 1% goat serum PBS solution, Molecular Probes) for 3 hrs at room temperature. Following final washes with 1% goat serum PBS solution, the neuronal cells on the coverslips were cover-slipped with a glycerol-based anti-photobleach medium. The cells were viewed under a confocal microscope (Carl Zeiss). Images were captured with a CCD camera and acquired by the Scion Image software (Scion Corporation, Frederick, MD)."}