Aif antibody

SH-SY5Y cells were grown on coverslips. After 24 h they were transiently transfected with the pEGFP-N1 SPG7^WT and mutant constructs with the use of Lipofectamine 3000 (ThermoFisher) following the manufacturer’s instructions. After 48 h, cells were fixed for 10 min at RT with 4% w/v paraformaldehyde in PBS, permeabilised with Methanol for 10 min at −20°C and quenched for 10 min at RT with 50 mM NH₄Cl in PBS. Cell mitochondria were stained using the AIF antibody (Cell Signaling Technology, Leiden, Netherlands) and nuclei using Hoechst 33342 (ThermoFisher). Coverslips were mounted with Dako Fluorescent Mounting Medium (Agilent, California, United States). Images were captured with a Zeiss fluorescent microscope using the Axiovision software. Colocalization index was quantified by Manders’ coefficient using Coloc2 plugin for ImageJ, and two-tailed t test was used to assess the p value. Mitochondrial morphology parameters were assessed with the use of a macro titled “mitochondrial morphology,” developed by the Chu Lab for ImageJ as previously described (Dagda et al., 2009 (link)). One-way ANOVA analysis was conducted for the statistical evaluation of the mitochondrial morphology parameters.