Immunofluorescence Analysis of Autophagy Markers

Immunofluorescence analysis was performed as described previously (Carroll et al, 2018 (link)). In brief, cells seeded on coverslips in 24‐well plates were fixed in 3.7% formaldehyde in PBS for 7 min at room temperature and permeabilised in methanol for 4 min at −20°C. Cells were then blocked for 1 h in 5% normal goat serum (Sigma‐Aldrich) in PBS at room temperature and incubated with primary antibodies overnight at 4°C. Cells were washed three times and incubated with the appropriate secondary antibodies for 1 h at room temperature (Thermo Fisher Scientific, A31556 and A21235, 1:1,000). Cells were washed, and coverslips were mounted on slides with fluoroshield mounting medium (Abcam). Fluorescence images were obtained described as above. The number of puncta colocalised with NDP52 or with YFP‐Parkin per cell was quantified. The following primary antibodies were used: mouse α‐Flag (Sigma‐Aldrich, F3165, 1:1,000, for NDP52 staining), rabbit α‐LC3 (CST, 3868S, 1:250), rabbit α‐Atg13 (CST, 13468S, 1:100) and rabbit α‐Atg16L (CST, 8089S, 1:100).