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The protocol provides a standardized method to assess the absolute number of NE neurons in the mouse LC using unbiased stereological counting. It covers brain preparation, immunostaining, delineation of the region of interest, image acquisition, neuron counting, and the use of Stereo Investigator software for workflow execution.",{"@graph":69,"@context":122},[70,84,105],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":40,"@type":76,"position":81},"https://docshare.wps.com/document/research-report/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/protocol-to-count-the-number-of-noradrenergic-neurons-in-mouse-locus-coeruleus-by-unbiased-stereology/432337/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/protocol-to-count-the-number-of-noradrenergic-neurons-in-mouse-locus-coeruleus-by-unbiased-stereology/432337.png","ImageObject",300,407,{"name":92,"@type":93},"Mimi","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-09-30","2026-09-29",true,{"@type":102,"interactionType":103,"userInteractionCount":8},"InteractionCounter",{"@type":104},"ViewAction",{"@type":106,"mainEntity":107},"FAQPage",[108,114,118],{"name":109,"@type":110,"acceptedAnswer":111},"What is the purpose of this protocol?","Question",{"text":112,"@type":113},"It provides a workflow to quantify the absolute number of noradrenergic neurons in the mouse locus coeruleus using unbiased stereological counting.","Answer",{"name":115,"@type":110,"acceptedAnswer":116},"Which main experimental steps does the protocol include?",{"text":117,"@type":113},"It outlines procedures for brain slice preparation, delineating the region of interest, immunostaining, image acquisition, and counting using Stereo Investigator software.",{"name":119,"@type":110,"acceptedAnswer":120},"Is the protocol limited to one mouse strain and age?",{"text":121,"@type":113},"The protocol has been applied to C57BL6/N mice at 1, 6, and 12 months and is also stated to be applicable to other mouse strains and disease models.","https://schema.org",{"og:url":83,"og:type":124,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":126,"canonical":83},"index,follow",{"doc_id":128,"site_id":62},432337,1790792210,{"code":4,"msg":5,"data":131},{"doc_id":128,"user_id":132,"nickname":92,"user_avatar":133,"doc_module":4,"category_id":39,"category_name":40,"doc_title":65,"doc_description":67,"doc_content":134,"file_id":135,"file_url":136,"file_type":137,"file_size":138,"view_count":8,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":139,"language":140,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":141,"faqs":142,"seo_title":143,"seo_description":67,"update_tm":144,"read_time":145},2336477974920,"https://ap-avatar.wpscdn.com/davatar_155a257f0dc6eb9ab79c44ca47cae57d","ll  \nOPEN ACCESS  \nProtocol  \nProtocol to count the number of noradrenergic neurons in mouse locus coeruleus by unbiased stereology  \nLetizia Zanetti, Maria Regoni, Elena Monzani,   , Michele Morari, Flavia Valtorta, Jenny Sassone  \nlz3041@cumc. columbia. edu (L.Z.)  \n[sassone.jenny@hsr.it](sassone.jenny@hsr.it) (J. S.)  \nHighlights  \nProtocol for assessing the absolute number of neurons in the mouse locus coeruleus  \nSteps for brain preparation, immunostaining, image acquisition, and counting  \nInstructions for using the Stereo Investigator software  \nZanetti et al. , STAR Protocols 7, 104273  \nMarch 20, 2026 © 2025 The Authors. Published by Elsevier Inc.  \n[https://doi.org/10.1016/](https://doi.org/10.1016/)[ ](https://doi.org/10.1016/)[j.xpro.2025.104273](j.xpro.2025.104273)  \nll  \nOPEN ACCESS  \nProtocol  \nProtocol to count the number of noradrenergic neurons in mouse locus coeruleus by unbiased stereology  \nLetizia Zanetti,1,2,3,6,* Maria Regoni,1,2 Elena Monzani,1 Cecilia Mini, 1,2,4 Giulia Di Giorgio,1 Michele Morari,5 Flavia Valtorta,1,2 and Jenny Sassone1,2,7,*  \n1Division of Neuroscience, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy  \n2Vita-Salute San Raffaele University, 20132 Milan, Italy  \n3Department of Psychiatry, Columbia University Irving Medical Center, New York, NY 10032, USA  \n4Department of Statistics, Computer Science, Applications (DiSIA), University of Florence, 50134 Florence, Italy  \n5Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131 Padova, Italy  \n6Technical contact  \n7Lead contact  \n*Correspondence: [lz3041@cumc.columbia.edu](lz3041@cumc.columbia.edu) (L.Z.), [sassone.jenny@hsr.it](sassone.jenny@hsr.it) (J.S.) [https://doi.org/10.1016/j.xpro.2025.104273](https://doi.org/10.1016/j.xpro.2025.104273)  \nSUMMARY  \nLoss of noradrenergic (NE) neurons in the locus coeruleus (LC) occurs in both Alzheimer’s disease (AD) and Parkinson’s disease (PD): pharmacological therapies aimed at neuroprotection in brain area are needed. Here, we present a protocol for assessing the absolute number of NE neurons in the mouse LC by unbiased stereological counting. We describe steps for preparing the brain slices, delineating the region of interest, counting the neurons, and using the Stereo  \nInvestigator software.  \nFor complete details on the use and execution of this protocol, please refer to  \nRegoni et al.1  \nBEFORE YOU BEGIN  \nThis protocol details the specific procedures for unbiased stereological counting of absolute numbers of noradrenergic (NE) neurons in the locus coeruleus (LC) of mice. We have successfully applied this protocol to C57BL6/N mice at 1, 6, and 12 months of age.1  \nImportantly, this protocol is also applicable to other mouse strains and disease models. This makes ita valuable tool for investigating LC degeneration associated with aging or diverse pathological conditions, and for assessing neuroprotective strategies.  \nInnovation  \nThe LC is a minute brain structure, which presents difficulties in access, sampling, and analysis, particularly in murine models. Standardized quantitative methods for its NE population are currently lacking in the literature. To assess the efficacy of neuroprotective therapies, a precise quantitative method is essential. For the first time, this protocol introduces a systematic workflow for sampling the LC, performing staining procedures, and achieving unbiased quantification of absolute NE neuron numbers via Stereo Investigator software. This protocol represents an important advance in the study of the LC at the preclinical level.  \nInstitutional permissions  \nAll experiments were designed to minimize the number of euthanized animals, in compliance with the 3Rs (replacement, reduction, and refinement) principle. Mice were housed and bred at the  \nSTAR Protocols 7, 104273, March 20, 2026 © 2025 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC license ([http://creativecommons.org","cbCaijeJIxbmthJx","https://ap.wps.com/l/cbCaijeJIxbmthJx","pdf",8941922,18,"English","# Highlights\n## Summary\n# Before you begin\n# Innovation\n# Institutional permissions\n# Key resources table","[{\"question\":\"What is the purpose of this protocol?\",\"answer\":\"It provides a workflow to quantify the absolute number of noradrenergic neurons in the mouse locus coeruleus using unbiased stereological counting.\"},{\"question\":\"Which main experimental steps does the protocol include?\",\"answer\":\"It outlines procedures for brain slice preparation, delineating the region of interest, immunostaining, image acquisition, and counting using Stereo Investigator software.\"},{\"question\":\"Is the protocol limited to one mouse strain and age?\",\"answer\":\"The protocol has been applied to C57BL6/N mice at 1, 6, and 12 months and is also stated to be applicable to other mouse strains and disease models.\"}]","Protocol to count the number of noradrenergic neurons in mouse locus coeruleus by unbiased stereology | PDF",1790659185,45]