Nanozoomer digital pathology system ndpview

Tissues were fixed in formalin, treated with concentrated formic acid to inactivate prions and embedded in paraffin. Tissue sections were subjected to deparaffinization through graded alcohols and heat-induced antigen retrieval in EDTA-based buffers. Stainings were performed on a NEXES immunohistochemistry robot (Ventana instruments) or on a BOND-III robot (Leica) using the following antibodies: 1:200 dilution of 3F4 (stock concentration 2 mg/ml, purified in house) for PrP staining; 1:3000 dilution of 4G8 (Signet) for Aβ staining; 1:1000 dilution of AT8 (Thermo Fisher Scientific) for staining of phosphorylated tau; 1:200 dilution of HPA040442 (Sigma) for cystatin F; 1:1000 dilution of rabbit anti-Iba1 (Wako). Immunoreactivity was visualized using an IVIEW DAB Detection Kit (Ventana) or Bond Polymer Refine Detection Kit (Leica). Haematoxylin and eosin staining was performed according to a standard protocol. For image analysis and acquisition, slides were scanned with NanoZoomer and images were obtained using the NanoZoomer Digital Pathology System (NDPview, Hamamatsu Photonics).

Stainings were performed on 2 µm sections from formalin fixed, formic acid treated, paraffin embedded tissues. After deparaffinization through graded alcohols, heat-induced antigen retrieval was performed in EDTA-based buffer CC1 and stainings were performed on a NEXES immunohistochemistry robot (Ventana instruments) using the following antibodies: Iba1 (1:1000, Wako); GFAP (1:13000, Dako); SAF84 (1:200, SPI bio). Only for the latter staining, antibody incubation was preceded by incubation with protease 2 (Ventana). Immunoreactivity was visualized using an IVIEW DAB Detection Kit (Ventana). Haematoxylin and eosin staining was performed according to standard procedures. Slides were scanned with NanoZoomer and images were visualized using the NanoZoomer Digital Pathology System (NDPview, Hamamatsu Photonics).

Histological analyses were performed on 2-μm thick sections from formalin fixed, formic acid treated, paraffin embedded brain tissues, as previously described[32 (link)]. Sections were subjected to deparaffinization through graded alcohols, followed by heat-induced antigen retrieval performed in EDTA-based buffer CC1 (Ventana). Stainings were performed on a NEXES immunohistochemistry robot (Ventana instruments) with the following antibodies: Iba1 (1:1000, Wako); GFAP (1:13000, Dako); SAF84 (1:200, SPI bio). For the latter staining, incubation with protease 2 (Ventana) was performed before antibody incubation. Immunoreactivity was visualized using an IVIEW DAB Detection Kit (Ventana). Haematoxylin and eosin stainings were performed according to standard procedures. Slides were scanned with NanoZoomer and images were acquired using the NanoZoomer Digital Pathology System (NDPview, Hamamatsu Photonics). High magnification pictures were quantified with an in-house developed python script by counting the number of HRP pixels, which were defined as pixels for which the following condition in RGB colour space holds true: R > G > B.