Caitlin A Finney1, Nicole M Jones1, Margaret J Morris2. 1. School of Medical Sciences, University of New South Wales, Sydney Australia. 2. School of Medical Sciences, University of New South Wales, Sydney Australia. Electronic address: m.morris@unsw.edu.au.
Abstract
BACKGROUND: Astrocytes play a critical role in CNS functions by providing physiological support to surrounding cells. These cells present a particularly unique challenge for in vitro immunohistochemical quantification due reactive gliosis after insult or injury, which is characterized by the extension of long processes. NEW METHOD: We present an optimized QuPath protocol that is scalable, fully automated, and capable of being applied to images generated by whole slide scanning technology using this open-source software. RESULTS: We induced mechanical injury in the rat brain and stained astrocytes using glial fibrillary acidic protein (GFAP) and 3,3-diaminobenzidine (DAB) chromogen detection. Slides were scanned using a whole slide scanner, Vectra Polaris. Using QuPath, we summarize and contrast three ways of quantifying astrocytes in uninjured (contralateral) and injured (ipsilateral) hemispheres: optical density, positive pixels and positive proportion. COMPARISON WITH EXISTING METHODS: Robust quantification of DAB stained astrocytes remains elusive. Previous methodologies have relied on software that is not compatible with whole slide scanner images. Use of such software can compromise the data integrity within the image and is limited by issues with scalability and lack of automation. Previous methods using manual histopathological scoring are also limited by the ability to quantify large numbers of astrocytes. Given these limitations, we were unable to directly compare our method with those using other software or manual histopathology. CONCLUSIONS: Based on an analysis of our method, we conclude that positive proportion may be the most effective way to quantify astrocytic responses using GFAP and DAB immunohistochemistry in the brain.
BACKGROUND: Astrocytes play a critical role in CNS functions by providing physiological support to surrounding cells. These cells present a particularly unique challenge for in vitro immunohistochemical quantification due reactive gliosis after insult or injury, which is characterized by the extension of long processes. NEW METHOD: We present an optimized QuPath protocol that is scalable, fully automated, and capable of being applied to images generated by whole slide scanning technology using this open-source software. RESULTS: We induced mechanical injury in the rat brain and stained astrocytes using glial fibrillary acidic protein (GFAP) and 3,3-diaminobenzidine (DAB) chromogen detection. Slides were scanned using a whole slide scanner, Vectra Polaris. Using QuPath, we summarize and contrast three ways of quantifying astrocytes in uninjured (contralateral) and injured (ipsilateral) hemispheres: optical density, positive pixels and positive proportion. COMPARISON WITH EXISTING METHODS: Robust quantification of DAB stained astrocytes remains elusive. Previous methodologies have relied on software that is not compatible with whole slide scanner images. Use of such software can compromise the data integrity within the image and is limited by issues with scalability and lack of automation. Previous methods using manual histopathological scoring are also limited by the ability to quantify large numbers of astrocytes. Given these limitations, we were unable to directly compare our method with those using other software or manual histopathology. CONCLUSIONS: Based on an analysis of our method, we conclude that positive proportion may be the most effective way to quantify astrocytic responses using GFAP and DAB immunohistochemistry in the brain.