Iview dab

Immunohistochemistry of c-Myc was conducted by the University of Colorado Cancer Center Histology Shared Resource. Five-micron-thick paraffin sections were deparaffinized, antigen unmasked, and immunohistochemically stained for c-Myc (Abcam, Cambridge, MA; rabbit monoclonal Y69; Cat# ab32072; dilution 1:50 in TBST + 1% BSA w/v). Antigen was revealed in pH 9.5 BORG solution (Biocare Medical, Concord, CA) for 10 min at 110 °C (NxGen Decloaking chamber, Biocare) with a 10-min ambient cool down. Immunodetection was performed on the Benchmark XT autostainer (Ventana Medical Systems, Tucson, AZ) at an operating temperature of 37 °C. Primary antibody was incubated for 32 min and detected with a modified I-VIEW DAB (Ventana) detection kit. I-VIEW secondary antibody and enzyme dispensers were replaced with full-strength and half-strength (diluted in PBS, pH 7.6) polymers, respectively (Rabbit ImmPress, Vector Laboratories, Burlingame, CA: cat# MP-7401). All sections were counterstained in Harris hematoxylin for 2 min, blued in 1% ammonium hydroxide (v/v), dehydrated in graded alcohols, cleared in xylene, and coverglass mounted using synthetic resin. Sections were examined through bright-field microscopy and c-Myc positive regions were quantified using ImageJ software.

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)}.