Anti neun

The histological analysis and immunohistochemistry were performed as previously described [76 (link), 77 (link)]. Briefly, female mice were perfused through the left cardiac ventricle with PBS, followed by 10% formalin under anesthesia. The brains were embedded in paraffin prior to a 48-h postfixation in 10% formalin. The paraffin blocks were sectioned at 3 μm, mounted onto glass slides, and stained with hematoxylin. For the immunofluorescence analysis, immunofluorescence was performed as previously described [78 (link)]. The antigen-retrieved slides were incubated in blocking buffer for 1 h at 25°C and treated with an anti-TDAG51 phycoerythrin (PE)-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), an anti-glial fibrillary acidic protein (GFAP, a marker of astrocytes and neoplastic cells of glial origin) Alexa Fluor 488-conjugated antibody (Santa Cruz Biotechnology) and anti-NeuN (a neuron-specific nuclear protein) Alexa Fluor 405-conjugated antibody (Novus Biological, Littleton, CO, USA) at 1:100 dilutions in blocking buffer for 16 h at 4°C. Finally, the stained slides were analyzed under an Olympus BX61 microscope (Olympus, Tokyo, Japan).

Samples were separated by 10% SDS-PAGE, transferred to nitrocellulose, blocked in 5% skimmed milk for 2 hrs and incubated with primary antibody for 1hr followed by incubation with secondary antibody (1:5000 dilution) for 30 min at room temperature. The chemiluminescent signal was detected via the enzymatic reaction using ECL detection reagents (Amersham) and visualized on ChemiDoc (Biorad). For quantitative immunodetection, the blots were incubated with a fluorescent tag (alexa488) secondary antibody for 30 min at room temperature. The primary antibodies used included anti-ATP synthase subunit β (MitoSciences MS503, 1:1000); anti-synaptophysin (Sigma, 1:1000); anti-tubulin (DSHB, 1:1000) and anti neuN (Novus Biologicals, 1:1000).