Fixation and imaging of HeLa cells after mitochondrial depolarization

Ectopic expression of p62/SQSTM1 often induces puncta formation and poor cell health due to p62’s proclivity to multimerize and form filaments. We used the cell fixation protocol described here in order to over-express fluorescently tagged p62 at low levels in p62-/- cells and amplify the signal with immuno-labeling. By this method, we were able to express a variety of tagged p62 mutations to determine their effects on NEMO recruitment to depolarized mitochondria without introducing toxic artifacts of p62 overexpression. We also note the use of a modified fixation protocol to visualize various endogenous ATG8 proteins, which are also difficult to over-express in our system. Thus, fixation techniques are a critical complement to studies in live cells.