Scanscope cs scanning system

To assess the relative intensity of striatal M1 and M4 immunostaining, M1- and M4-immunostained sections were scanned at 20× using a ScanScope CS scanning system (Aperio Technologies, Vista, CA, USA), and digital images of the stained tissue slides containing different levels of striatum (rostral, middle and caudal) were obtained by ImageScope viewer software (Aperio) and imported into ImageJ (National Institutes of Health, Schneider et al., 2012 (link)) for optical density measurement. The images were converted into 16-bit grayscale format and inverted. To control for differences in background staining, the optical density measurement in the corpus callosum (cc) was subtracted from that obtained in striatal measurements. Measurements of the intensity of labeling were obtained in three sections at rostral, middle and caudal striatal levels from DRD (n = 3) and WT (n = 3) animals in the 3-month old group. In each striatal section, the intensity of M1 and M4 immunostaining was measured in all quadrants delineated in rostral, middle and post-commissural striatum (see Figure 1). The resulting intensity values were averaged in DRD and WT group for comparison.

This protocol was approved by the University Health Network Institutional Review Board. Twelve NASH patients were included for HNF4A IHC validation using anti HNF4-Alpha, mouse monoclonal antibody (Abcam, ab201460, rabbit monoclonal, clone EPR16885-99). FFPE sections were (5um) pre-treated for antigen retrieval following manufacturer’s instruction. The dilution for HNF4-alpha antibody was 1:2000 and an anti-rabbit was used as the secondary antibody. The complex was then visualized with hydrogen peroxide substrate and 3, 3’- diaminobenzidine tetrahydrochloride (DAB) chromogen. The slides were then counterstained with Harris Hematoxylin. The degree of HNF4-Alpha staining in each NASH case and healthy liver (control) was assessed using Imagescope software. Entire slides were digitally scanned by an Aperio ScanScope CS scanning system and analyzed by Aperio ImageScope Viewer software (Aperio Technologies Inc., Vista, CA) using the Positive Pixel Count v9 algorithm.