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However, post-processing of photomicrographs acquired after 3,3\u2032-Diaminobenzidine (DAB) staining is particularly challenging due to the complexity associated with the size, samples number, analyzed targets, image quality, and even the subjectivity inherent to the analysis by different users. Conventionally, this analysis relies on the manual quantification of distinct parameters (e.g., the number and size of cells and the number and length of cell branching) in a large set of images. These represent extremely time-consuming and complex tasks, defaulting the processing of high amounts of information. Here we describe an improved semi-automatic method to quantify glial fibrillary acidic protein (GFAP)-labelled astrocytes in immunohistochemistry images of rat brains, at magnifications as low as 20\u00d7. This method is a straightforward adaptation of the Young &amp; Morrison method, using ImageJ\u2019s plugin Skeletonize, coupled with intuitive data processing in datasheet-based software. It allows swifter and more efficient post-processing of brain tissue samples, regarding astrocyte size and number quantification, the total area occupied, as well as astrocyte branching and branch length (indicators of astrocyte activation), thus contributing to better understand the possible inflammatory response developed by astrocytes.<\/jats:p>","DOI":"10.3390\/ijms24054508","type":"journal-article","created":{"date-parts":[[2023,2,27]],"date-time":"2023-02-27T03:23:36Z","timestamp":1677468216000},"page":"4508","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["A Semi-Automatic Method for the Quantification of Astrocyte Number and Branching in Bulk Immunohistochemistry Images"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8090-5720","authenticated-orcid":false,"given":"Sandra I.","family":"Marques","sequence":"first","affiliation":[{"name":"UCIBIO\u2014Applied Molecular Biosciences Unit, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal"},{"name":"i4HB\u2014Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6650-5285","authenticated-orcid":false,"given":"Helena","family":"Carmo","sequence":"additional","affiliation":[{"name":"UCIBIO\u2014Applied Molecular Biosciences Unit, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal"},{"name":"i4HB\u2014Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3858-3494","authenticated-orcid":false,"given":"F\u00e9lix","family":"Carvalho","sequence":"additional","affiliation":[{"name":"UCIBIO\u2014Applied Molecular Biosciences Unit, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal"},{"name":"i4HB\u2014Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5078-980X","authenticated-orcid":false,"given":"Susana I.","family":"S\u00e1","sequence":"additional","affiliation":[{"name":"Unit of Anatomy, Department of Biomedicine, Faculty of Medicine, University of Porto, Alameda Prof. Hern\u00e2ni Monteiro, 4200-319 Porto, Portugal"},{"name":"CINTESIS@RISE, Faculty of Medicine, University of Porto, Alameda Prof. Hern\u00e2ni Monteiro, 4200-319 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5656-0897","authenticated-orcid":false,"given":"Jo\u00e3o Pedro","family":"Silva","sequence":"additional","affiliation":[{"name":"UCIBIO\u2014Applied Molecular Biosciences Unit, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal"},{"name":"i4HB\u2014Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"430","DOI":"10.1016\/j.neuron.2008.10.013","article-title":"The mystery and magic of glia: A perspective on their roles in health and disease","volume":"60","author":"Barres","year":"2008","journal-title":"Neuron"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.neulet.2013.12.071","article-title":"The dual role of astrocyte activation and reactive gliosis","volume":"565","author":"Pekny","year":"2014","journal-title":"Neurosci. 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Toxicol."},{"key":"ref_7","first-page":"387","article-title":"Dendritic organization in the neurons of the visual and motor cortices of the cat","volume":"87","author":"Sholl","year":"1953","journal-title":"J. Anat."},{"key":"ref_8","first-page":"e57648","article-title":"Quantifying microglia morphology from photomicrographs of immunohistochemistry prepared tissue using ImageJ","volume":"5","author":"Young","year":"2018","journal-title":"J. Vis. Exp."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"632","DOI":"10.1016\/j.trstmh.2012.05.004","article-title":"Application of ImageJ program to the enumeration of Orientia tsutsugamushi organisms cultured in vitro","volume":"106","author":"Siritantikorn","year":"2012","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_10","first-page":"428","article-title":"Astrocyte intermediate filaments in CNS pathologies and regeneration","volume":"204","author":"Pekny","year":"2004","journal-title":"J. Pathol. J. Pathol. Soc. 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