Immunohistochemistry

Mouse brains were fixed in 4% paraformaldehyde for 12 h. Paraffin sections (thickness, 5 μm) were de-paraffinized in xylene, re-hydrated, dipped in 0.01 M citrate buffer (pH 6.0), and microwaved at 120 °C for 15 min. After blocking with 10% FBS containing PBS, sections were incubated with primary antibody for 12 h at 4 °C, washed with PBS three times at RT, and incubated with secondary antibodies at RT for 1 h. All procedures were performed in parallel for all mouse groups being compared.

The antibodies used for immunohistochemistry were diluted as follows: rabbit anti-calbindin D-28K antibody, 1:2000 (C2724, Sigma); mouse anti-calbindin D-28K antibody, 1:2000 (C9848, Sigma); rabbit anti-YAP antibody, 1:100 (#sc-15407, Santa Cruz Biotechnology); rabbit anti-YAPdeltaC antibody, 1:1000 (raised against the common COOH-terminal peptide [SVFSRDDSGIEDNDNQ]); rabbit anti-RORα, 1:100 (#sc-28612, Santa Cruz Biotechnology); rabbit anti-Tip60, 1:100 (#sc-25378, Santa Cruz Biotechnology); mouse anti-Atxn1, 1:100 (#MABN37, Millipore); anti-γH2AX, 1:100 (#05-636, Millipore); rabbit anti-53BP1, 1:5000 (NB100-304, Novus Biologicals); mouse anti-RpA1, 1:100 (H-7, Santa Cruz Biotechnology, CA, USA); Cy3-conjugated anti-rabbit IgG, 1:500 (711-165-152, Jackson Laboratory, Bar Harbor, ME, USA); Alexa Fluor 488–conjugated anti-mouse IgG, 1:1000 (A21202, Molecular Probes, Eugene, OR, USA); Cy3-conjugated anti-mouse IgG, 1:500 (715-165-150, Jackson Laboratory); Alexa Fluor 488-conjugated anti-rabbit IgG, 1:1000 (A21206, Molecular Probes).