Rabbit anti lc3

Rabbit anti-human GCDH antibody was kindly provided by Dr. S. I. Goodman (University of Colorado Health Sciences Center, Denver). The polyclonal mouse anti-human DLST and rabbit anti-human ETFA antibodies were purchased from Sigma (Munich, Germany), rabbit anti-human ETFB from Abcam (Cambridge, UK), and rabbit anti-LC3 from Abgent (San Diego, USA). The monoclonal mouse anti-GFP antibody was obtained from Roche (Mannheim, Germany) and rabbit anti-MnSOD from Millipore (Billerica, USA). Peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG was from Dianova (Hamburg, Germany). HRP-conjugated anti-V5 antibody, monkey anti-mouse IgG coupled to Alexa Fluor 488 and goat anti-rabbit IgG coupled to Alexa Fluor 546 were from Invitrogen (Karlsruhe, Germany).

Briefly, paraffin-embedded sections were deparaffinized and washed three times with PBS for 5 min. Subsequently, the sections were immersed in EDTA-Tris solution (pH 9.0) for 30 min at 98 °C for antigen retrieval and rinsed three times with PBS for 5 min. Then, the slides were incubated with 10% non-immune goat serum for 30 min at room temperature to block non-specific staining. After this, the slides were incubated with primary antibodies: rabbit anti-LC3 (1:100, Abgent) and rabbit anti-LAMP1 (1:200, Cell Signaling Technology) in humidified chambers at 4 °C overnight. The next day, after washing these sections in PBS, isothiocyanate (TRITC)-conjugated anti-rabbit secondary antibodies (1:200, Santa Cruz) were then applied for 1 h at 37 °C. All stained specimens were observed under a confocal laser scanning microscope (Leica, Wetzlar, Germany). For the quantitative analysis, the average score of six randomly selected areas (three slides for each brain) was calculated using National Institutes of Health (NIH) Image Pro Plus 6.0 software.