Ab71558

A polyclonal antibody against the excitatory amino acid transporter 3 (EAAT3) was purchased from Santa Cruz Biotechnology (#sc-25658, Santa Cruz, TX, United States). Polyclonal antisera obtained from Chemicon (Temecula, CA, United States) were used for detection of glutamate-aspartate transporter EAAT1 (GLAST, #AB1782) and glial glutamate transporter EAAT2 (GLT-1, #AB1783). Protein kinase C-alpha (PKCα) was detected by a monoclonal antibody obtained from BD Biosciences (#610107, Heidelberg, Germany). A polyclonal antibody against actin was purchased from SIGMA (#A5060 St. Louis, MO, United States). Morphology of hippocampal neurons was visualized by a polyclonal antiserum against microtubule associated protein 2 (MAP2, #AB5622) and neurofilament protein of 200 kDa (#AB5256) from Chemicon International (Hofheim, Germany). An affinity purified polyclonal rabbit IgG against full length C3bot developed by our group was applied (Rohrbeck et al., 2014 (link)). To detect the phosphorylation levels of EAAT3 a polyclonal antibody directed against phosphotyrosine was purchased from Santa Cruz Biotechnology (#sc-7020). Antibodies against protein kinase C isoforms γ (monoclonal #ab71558), ε (polyclonal #ab63638), and ζ (polyclonal #ab108970) were obtained from Abcam (Cambridge, United Kingdom).

For histological analysis of the cerebellum, anesthetized mice (ketamine/xylazine: 0.1 ml/10 g) were fixed by transcardial perfusion using 4% PFA. For further fixation, brains were incubated overnight in 4% PFA at 4°C and processed as described in standard protocols for cryo (15 μm sections) and paraffin sectioning (5 μm sections). For immunofluorescent staining of cryo-sections, following primary antibodies were used: anti-calbindin-D28k (Swant; CB300), anti-PKCγ (Abcam; ab71558). Calbindin-D28k immunohistological stainings were performed as described in Storbeck et al. (2014) (link) using an ABC kit (Vector Laboratories). To acquire the whole Purkinje neuron cell body, different number of stacks were included in the analysis. All samples were imaged simultaneously with the same settings. The experimentator was blinded at all steps to exclude any bias during imaging.