Figure 3.

(A) Flowchart of image acquisition through processing to acquiring the final counts. Images are acquired after immunohistochemistry and are loaded into CellProfiler before undergoing background removal. A conceptual overview of image processing is presented. (B). Images are processed through several routes, depending on which stain is in the image. For example, Hoechst is used to identify nuclei objects before being associated with a phenotype stain (GFAP) to identify a phenotype population (astrocytes). This phenotype population is then masked with the binary version of the ECM molecule (TSP) which has gone through background removal, to identify the amount of that phenotype population that associates with the ECM molecule (astrocytes associating with TSP). There may be double checks, if possible, to exclude incorrectly identified objects. For astrocytes, identified astrocyte nuclei were masked with the stain for neurons (NeuN) to ensure that no identified objects were also being identified as neurons. The final counts were then exported. Abbreviations: ECM, extracellular matrix; GFAP, glial fibrially acid protein; TSP1, thrombospondin 1.