Tris borate edta buffer

Immunohistochemical staining of xenograft tumors was done on formalin-fixed and paraffin embedded 4-μm sections using the Ventana autostainer model Discover XT (Ventana Medical System, Tucson, AZ) with an enzyme-labeled biotin streptavidin system and solvent-resistant 3,3`-diaminobenzidine Map kit. Slides were pretreated with tris borate EDTA buffer (pH 7.8, Roche) for 48 minutes. The following antibodies were used: Ki-67 (mouse monoclonal antibody, DAKO, Denmark), and cleaved caspase-3 (rabbit polyclonal antibody, Cell Signaling). Specificity of staining was controlled by including an unspecific control antibody (DAKO). Slides were counterstained with hematoxylin (Roche). Expression was evaluated manually. Number of Ki-67/cleaved caspase-expressing cells was determined by counting the number of positive cells. The numbers stated in the graphs are means of 10 visual fields at a magnification of 200-fold.

Cases and controls brain samples were derived from the Mount Sinai Neuropathology Brain Bank. Inclusion criteria were individuals with a neuropathological diagnosis of Parkinson’s disease for cases and cognitively normal with no or only age-related neuropathological changes for controls. Formalin-fixed paraffin-embedded (FFPE) sections (5μm) were prepared from substantia nigra blocks, mounted on positively charged slides, and baked overnight at 70 °C. Immunohistochemistry (IHC) was performed on a Ventana Benchmark XT automatic staining platform (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s protocol with reagents and antibodies acquired from the same lot. Antigen retrieval was done using CC1 buffer (Tris/Borate/EDTA buffer, pH 8.0–8.5, Roche Diagnostics) for 1 h followed by primary antibody incubation. All primary antibodies were diluted in antibody dilution buffer (ABD24, Roche Diagnostics). Primary antibodies were incubated for 36 min (mOXDJ1, 1:400, Abcam) or 28 min (GFAP, 1:10, Ventana, 760-4345) followed by either 3,3’-diaminobenzidine (DAB) or alkaline phosphatase for visualization. For slides that were double-labeled, both DAB and alkaline phosphatase were used for visualization. All slides were counterstained with hematoxylin and coverslipped.