Ventana automated immunohistochemical staining machine

For the analysis of brain histopathology 15-week-old WT and Zfp106^−/− mice were perfused transcardially with saline followed with 4% PFA, brain transferred to formalin and embedded in paraffin wax. Staining with H&E and anti- GFAP, IBA-1, p62, MBP and neurofilament antibodies was undertaken using a Ventana automated immunohistochemical staining machine (Ventana Medical Systems, Tuscon, AZ, USA). Paraffin-embedded sections once incubated with appropriate secondary antibodies were developed using 3,30-diaminobenzidine and counterstained with haematoxylin.
Twenty micrometer transverse sections from the lumbar region of fixed spinal cords collected onto glass slides were immuno-fluorescently stained with anti-GFAP (Cy-conjugated mouse monoclonal, Sigma; used at 1:1000) and anti-IBA1 (rabbit polyclonal, Abcam; used at 1:500), visualized using a secondary AlexaFluor488-conjugated antibody (Invitrogen) and counterstained with NeuroTrace® 435⁄455 fluorescent Nissl stain (Invitrogen). A C-terminal p62 antibody (Progen, Germany; 1:300) was also used to stain lumbar spinal cord sections. Confocal images were taken using a Zeiss 710 microscope. Active cell death was analysed using TUNEL staining (Promega) following manufacturer recommendations.

Left brain hemispheres fixed in 10% buffered formol saline were assessed for presence of abnormal PrP by staining with the anti-PrP monoclonal antibody ICSM35 (D-Gen, London) using a Ventana automated immunohistochemical staining machine (Ventana Medical Systems, Tuscon, AZ, USA) as described previously^{48 (link)}.Tissue was fixed in 10% v/v buffered formol saline followed by incubation in 98% v/v formic acid for 1 h. Following further washing for 24 h in 10% v/v buffered formol saline tissue samples were processed and embedded in paraffin wax. Sections were cut at a nominal thickness of 4 µm, treated with 98% v/v formic acid for 5 min and the slides were placed on the automated staining machine. De-paraffinisation was performed with xylene followed by heating to 95 °C in a low ionic strength buffer (2.1 mM Tris, 1.3 mM EDTA, 1.1 mM sodium citrate, pH7.8) for 30 min, before 16 min protease treatment. Abnormal PrP accumulation was detected using anti-PrP monoclonal antibody ICSM35^{49 (link)} in conjunction with a biotinylated-anti-mouse IgG secondary antibody (iView SA-HRP, Ventana Medical Systems) before development with 3′3 diaminobenzidine tetrachloride as the chromogen (iView DAB, Ventana Medical Systems). Immunostaining for glial fibrillary acidic protein (GFAP) and haematoxylin and eosin (H&E) staining of serial sections was done according to published methods^{48 (link)}.