Fig. 2

Global phenotypic expression organizes CNS sub-regions in a data driven manner. a Schematic of brain regions used in construction of TMA; circle highlights 3 mm cores isolated from FFPE tissues. The photomicrograph of the brain TMA cores stained with Luxol Fast Blue/Hematoxylin and Eosin (LFB/H&E) and the rastered areas, highlighted in black boxes, are shown in Additional file 1: Fig. S2A. b Heatmap of mean z-score distribution of pixel expression of proteins per rastered FOV (row normalized). Columns and rows are hierarchically clustered (Euclidean distance). Variance between and among FOVs are stratified in an unsupervised manner into similar anatomical regions. Black boxes highlight distribution of different calcium binding proteins, PV predominantly expression in cerebellum, midbrain, medullar similar to previous reports in mouse and rat¹, while CB expression is enriched in stratum moleculare cerebellum layer highlighting presence of Purkinje cells, and CR in the LC c, d Tiled spectral images that were pseudo-colored to show distribution of calcium-binding protein PV, CB and CR (c) and TH and GAD65/67 (d). Enlargement of the boxed areas in c and d show that CB+ Purkinje neuron in CBL co-localized with PV, CR and VGAT, and TH+ dopaminergic neurons in SN and LC co-localized with GAD^65/67. Each brain region is composed of 4 tiled FOVs. Each FOV is a 500 µm² spectral image acquired at low-resolution scan resolving ~ 1 µm²/pixel. Abbreviations: CA1, Cornu Ammonis 1; STR, Striatum; SN, Substantia Nigra; LC, Locus Coeruleus; CBL, Cerebellum; MO, Medulla Oblongata; CB, CALBINDIN; CR, CALRETININ; PV, PARVALBUMIN; TH, Tyrosine Hydroxylase; VGAT, Vesicular GABA Transporter; GAD^65/67, Glutamate Decarboxylase 65/67