摘要
It has been reported that distal cerebrospinal fluidcontacting neurons（dCSF-CNs）can be detected by immunohistochemical assay using cholera toxin subunit B-conjugated horseradish peroxidase（CBHRP）.In the present study,another two methods with CB alone or CB-conjugated FITC（CB-FITC）were used,and the results from the three methods were compared.Adult Sprague-Dawley rats were randomly divided into three groups with CB-HRP,CB or CB-FITC.Tracers were diluted to 30%in artificial cerebrospinal fluid and injected separately（in a volume of 3μL）into the lateral ventricle.Animals from the CB-HRP and CB groups were perfused 48 h after surgery while animals from the CB-FITC group were perfused 1,3,6,12,24 or 48 h after surgery.The brain was sectioned（40μm）for immunofluorescence and five sections with positive neurons were selected from each rat for neuron counting.Three clusters of positive neurons in a＇Y-like＇distribution were detected ventral to the cerebral aqueduct of rats from the three groups.No significant difference was observed among the quantitative data.In the CB-FITC group,stable staining was detected even at 6 h after injection.Taken together,lateral ventricle injection of CB/CB-FITC is a useful method for labeling dCSFCNs in rats.The CB-FITC method makes dCSF-CNs labeling much simpler and more convenient.
It has been reported that distal cerebrospinal fluidcontacting neurons（dCSF-CNs）can be detected by immunohistochemical assay using cholera toxin subunit B-conjugated horseradish peroxidase（CBHRP）.In the present study,another two methods with CB alone or CB-conjugated FITC（CB-FITC）were used,and the results from the three methods were compared.Adult Sprague-Dawley rats were randomly divided into three groups with CB-HRP,CB or CB-FITC.Tracers were diluted to 30%in artificial cerebrospinal fluid and injected separately（in a volume of 3μL）into the lateral ventricle.Animals from the CB-HRP and CB groups were perfused 48 h after surgery while animals from the CB-FITC group were perfused 1,3,6,12,24 or 48 h after surgery.The brain was sectioned（40μm）for immunofluorescence and five sections with positive neurons were selected from each rat for neuron counting.Three clusters of positive neurons in a＇Y-like＇distribution were detected ventral to the cerebral aqueduct of rats from the three groups.No significant difference was observed among the quantitative data.In the CB-FITC group,stable staining was detected even at 6 h after injection.Taken together,lateral ventricle injection of CB/CB-FITC is a useful method for labeling dCSFCNs in rats.The CB-FITC method makes dCSF-CNs labeling much simpler and more convenient.
基金
supported by grants from the National Natural Science Foundation of China (30871307, 81200862)
the Fund of Science and Technology Innovation Team in Jiangsu Province, China (2009), Open Project Funds of the Jiangsu Key Laboratory of Brain Disease Bioinformation (Jsbl1107)
the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (12KJD320004)
