Calbindin

Wild-type and SCA 17 transgenic mice were cardiac perfused first with PBS and then fixed with 4% formaldehyde (EM grade) at the age of 24 weeks. Cerebellar specimens were then embedded in paraffin, cut into sections (5 μm), and placed on adhesion microscope slides (Thermo Fisher Scientific). By using the heat-induced epitope retrieval method, the sections were separately stained at room temperature for 1 hour with antibodies of calbindin (1:500; Cell Signaling Technology) or TBP (1:1,000; Santa Cruz Biotechnology Inc.). Detection was performed by incubation with biotinylated secondary antibodies (Novolink™ Polymer Detection System l; Leica Microsystems, Wetzlar, Germany), for 30 minutes at room temperature, followed by 30-minute incubation with avidin–biotin–HRP complex (Novolink™ Polymer Detection System l). Visualization was performed with 3,3′-diaminobenzidine Chromogen (Novolink™ Polymer Detection System l) and counterstained with hematoxylin (Novolink™ Polymer Detection System l) following the supplier’s protocol.

TMT was purchased from Fujifilm Wako Chemical Co. (Miyazaki, Japan). Fluoro-Jade B (FJB) was obtained from Nacalai Tesque (Kyoto, Japan). Western Lightning Chemiluminescence Reagent Plus was product of PerkinElmer (Waltham, MA). IgGs were purchased from companies below: calbindin and GFAP (Cell Signaling Technology Inc., Danvers, MA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and N-terminus of GABA_B receptor R2 (GABA_BR2) subunit (Santa Cruz Biotechnology, Santa Cruz, CA). C-terminal regions of GABA_BR1 and GABA_BR2 subunits were produced as previously described [8 (link)]. Secondary antibodies used were anti-rabbit IgG conjugated with Alexa Fluor® 555 (Cell Signaling Technology Inc., Danvers, MA), anti-mouse IgG conjugated with FITC antibody (Sigma Chemicals, St. Louis, MO) and with Alexa Fluor® 488 (Thermo Fisher Scientific Inc., Waltham, MA) for immunohistochemistry, or horseradish peroxidase conjugated anti-rabbit IgG (Dakocytomation Carpinteria, CA), anti-chicken IgY (Jackson ImmunoResearch Inc., West Grove, PA) and anti-mouse IgG (GE Healthcare Life Sciences, Little Chalfont, United Kingdom) for immunoblot analysis. All other chemicals were of the highest purity available.

Antibodies were purchased from SantaCruz Biotechnology (Dallas, TX, USA) (A2, Polyamine Oxidase), Sigma (St. Louis, MO, USA) (Calbindin, Tubulin) and Cell Signaling Technology (Boston, MA, USA) (FASL, P-JNK/SAPK, total JNK/SAPK, BID and cytochrome c).