The Staining Method And Expression Study Of Neurogenesis-related Markers In Aging Human Hippocampus

BACKGROUNDAdult Hippocampal Neurogenesis(AHN)is believed to be essential in spatial learning,pattern separation and the regulation of memory and emotion.AHN study will bring light to new treatment in neurodegenerative diseases including the Alzheimer's disease.However,whether AHN persists in aged people is still controversial,and the protocols of tissue processing and immunofluorescence staining require improvement.OBJECTIVEThis study aims to find out the proper protocol to carry out immunofluorescence staining of hippocampal neurogenesis markers utilizing post mortem tissues from National Human Brain Bank for Development and Function,and study the expression patterns of common neurogenesis-related markers in dentate gyrus of aged people.METHODSConduct brain extraction and hippocampus dissection on subjects,along with fixation,dehydration,embedding,ice-slicing and immunofluorescence staining.Compare the difference of the outcome using different fixatives,preservation methods,antigen-retrieval protocols,immunofluorescence staining steps and Sudan Black B quenching and so on.Take photographs of DCX/SOX2/Nestin positive cells to observe their features like morphology and distribution,and measure their lengths of nucleus.RESULTSImmediate fixation with fresh 4%PFA at 4?,following sucrose dehydration and-80?freezing is the best tissue processing protocol for protected structure.0.5%NaBH4 incubation and Heat-induced antigen retrieval with CB(pH=6)as well as incubation with 0.01%Sudan Black B in 70%ethanol are required for the antigenicity and minimal nonspecific fluorescence.DCX+cells of different morphologies are around GCL,SGZ together with ML of the dentate gyrus of aged people,some of which are co-labeled with SOX2 or NeuN.SOX2+ cells are also abundant in above areas.Nestin+ cells mostly located in inner GCL and SGZ,with many processes and lipofuscin in cytosol,and they can be divided in different subgroups according to nucleus position.Ascl-1+ cells are the most abundant and they almost seldom co-express with SOX2.CONCLUSIONThere are plenty of cells positive with hippocampal neurogenesis markers in the dentate gyrus of aged people with normal cognitive abilities.Cells positive with the same single markers are heterogeneous,suggesting they belong to different subgroups of various functions.In addition,this study comes up with a better protocol for human post-mortem tissue processing and immunofluorescence staining for hippocampal neurogenesis markers,which reduce nonspecific staining and autofluorescence to the most while protect the structure and antigenicity.