In IHC experiments, the mouse euthanized at 593 dpi had plaque-like PrPSc deposits in pons (Fig. 2a), and some of these deposits stained with Thioflavin S, indicating that they had amyloid properties. In the mouse euthanized at 601 dpi, perineuronal deposits were seen in pons (Fig. 2b) and coarse neuropil deposits were found in the thalamus (Fig. 2c). Gray matter vacuolation, astrogliosis and microgliosis, all typical of prion disease were also noted in the regions of PrPSc deposition (Fig. 2). Two additional mice euthanized at 716 and 718 dpi also had PrPSc deposits in most of these same brain regions (Table 2). Thus, detection of PrPSc by IHC correlated exactly with detection by immunoblotting.Fig. 2Immunohistochemistry and neuropathology of tg66 mice injected with Y226X human brain homogenate. Panel a Pons region of a mouse euthanized at 593 dpi. PrPSc was detected by IHC using biotinylated antibody 3F4 as described in the methods (panel a-1). Large and medium-sized PrPSc deposits are seen at higher magnification (a-2). Inset in a-2 shows Thioflavin S staining of one aggregate. Typical prion disease vacuolation (arrow) is shown by H&E staining (a-3), and astrogliosis and microgliosis (arrow) are shown by anti-GFAP staining (a-4) and anti-Iba1 staining (a-5). Panel b Pons region of a mouse euthanized at 601 dpi. PrPSc staining showed smaller coarse deposits (b-1), and perineuronal and linear axonal staining (arrows) could be seen at higher magnification (b-2). Vacuolation, astrogliosis and microgliosis (arrows) was also prominent in this same area (b-3, b-4, b-5). Panel c: Thalamus of same mouse shown in panel b showed slightly finer staining of PrPSc at both low (c-1) and high (c-2) magnification. Prominent vacuolation (c-3), astrogliosis (c-4) and microgliosis (c-5) was also noted (arrows). Scale bars shown in a-1, b-1 and c-1 are 200 μm, scale bars shown in a-2, b-2 and c-2 are 50 μm and apply to each subsequent panel within the same figure letterTable 2Characterization and location of PrPSc deposits in tg66 mice injected with Y226X human brain tissueBrain regionsaDpibPonsThalamusSuperior ColliculusHypothalamusCerebral cortex593PCCPneg601P&PNCCPNPN716P&PNnegnegPP (rare)718P&PNnegC (rare)negC (weak)a-Types of PrPSc deposits seen in the indicated brain regions: P = plaque-like, PN = perineuronal linear or pericellular deposits, C = coarse deposits in neuropil, neg = negativeb-Days post-injection with human Y226X brain tissue