A scalable, fully automated approach for regional quantification of immunohistochemical staining of astrocytes in the rat brain

Caitlin A. Finney, Nicole M. Jones, Margaret J. Morris*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Article number108994
Pages (from-to)1-10
Number of pages10
JournalJournal of Neuroscience Methods
Volume348
Early online date8 Nov 2020
DOIs
Publication statusPublished - 15 Jan 2021
Externally publishedYes

Keywords

  • Astrocytes
  • Immunohistochemical analysis
  • Immunohistochemical quantification
  • QuPath
  • Whole slide scanning

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