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However, questions have been raised whether DCX is expressed by cell types other than immature neurons 21, 29, may be re-expressed in de-differentiating mature neurons 30, or represent developmentally generated immature neurons 31. Short PMI and the use of 4% PFA are considered optimal for labile antigens such as PSA-NCAM and DCX 23, 28. Differences in tissue fixation, post-mortem interval (PMI), and staining protocols 23, may contribute to discrepant results. Stereology was applied in studies reporting either the absence 26, 27 or presence 22, 23, 24 of hippocampal neurogenesis. Analyses from gestation to old age show no new hippocampal neurons after the teenage years 25, or even sooner, from early childhood 26, 27. In contrast, three other recent studies question adult human hippocampal neurogenesis 25, 26, 27. In addition, three recent reports indicate there are large numbers of DCX + cells in adulthood 22, 23, 24, which are only reduced in Alzheimer’s Disease (AD) patients 23, 24. Histological staining using endogenous markers for cell cycle (e.g., Ki67, MCM2) and immature neurons (e.g., PSA-NCAM, DCX) 3, 4, 20, 21 provided corroborating evidence. The first evidence for human neurogenesis, obtained from post-mortem brain tissue from cancer patients treated with the thymidine analog bromodeoxyuridine (BrdU) 18, was further supported by research correlating atmospheric 14C released by nuclear bomb testing with the incorporation of 14C into genomic DNA of dividing cells 19. However, the extent and amount of neurogenesis in humans remains unclear. As such, enhancing adult neurogenesis may have therapeutic translational potential.
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Exercise is now considered one of the most effective interventions for delay or prevention of aging-related neurodegeneration 15, 16, 17. Running has a strong positive effect on adult neurogenesis 13, 14 in association with enhanced synaptic plasticity and memory function (see recent reviews refs. The functional relevance of new hippocampal neurons is based on regulatability by a variety of stimuli, including both behavioral and biological conditions 7, 12. Adult-born neurons exhibit enhanced synaptic and structural plasticity for weeks to months during their integration into neural circuits 2, 11. Around ~4 weeks of age the primary dendrite has extended multiple branches into the molecular layer, mature neuronal markers (NeuN, Calbindin) are expressed, axons have reached area CA3, and the cells receive afferent entorhinal cortex input. GABAergic neurotransmission becomes inhibitory at 2–3 weeks of new neuron age, concomitant with the growth of dendritic spines (reviewed by refs. One-week-old neuroblasts receive basal forebrain cholinergic as well as intrahippocampal excitatory glutamatergic and GABAergic inputs 9, 10. These cells are gradually incorporated into the hippocampal network. Ki-67, MCM2), that can develop into neuroblasts (type 3 cells), positive for doublecortin (DCX), PSA-NCAM, Calretinin (reviewed by refs. In particular, radial glial cells, called type 1 cells, are neural stem cells that give rise to rapidly amplifying progenitors (type 2 cells) expressing proliferation markers (e.g.
Unbiased stereology using optical fractiontor probe ki67 how to#
Detailed User Guide covering not only how to use software but also relevant stereology principles (Mouton 2002, 2011).Adult-born hippocampal neurons mature over weeks of sequential changes in morphology, physiology and stage-specific expression of molecular markers 2, 7.Consultation services regarding preferred histology and tissue preparation methods for accurate stereology.Integration with FIJI/ImageJ software to automatically stitch 2D captured images and create super-high-resolution images and automatically build 3D images stacks.Integrated Training Module to ensure consistent data collection across multiple investigators.Digital Stereology™ for automatic capture and manual, semi-automatic and fully automatic stereology analysis of tissue using unbiased methods (Mouton et al., 2017).Automatic workflow optimization to collect data with maximum efficiency (Alahmari et al., 2019).Virtual Cycloids™ for unbiased analysis of surface area and length, respectively, on arbitrary sections (Gokhale et al., 2004).Rare Event Protocol™ to standardize data collection of rare events, i.e., objects with extremely limited distribution (Mouton 2011).Space Balls™ for unbiased analysis of length/length density of curvilinear object, e.g., fibers, axons, dendrites, capillaries, etc.Current version of Stereologer software installed on a new computer for high-resolution computerized stereology, fully integrated with microscope and camera.