Mitochondria gfp

Prior to fixation, the cells were counterstained with CellMask Orange plasma stain (C10045) to identify the cell’s cytoplasmic area. The cells were washed and fixed with an equal amount of warm 4% paraformaldehyde (PF) in phosphate-buffered saline (PBS). To identify intra-cellular structures, the cells were transfected with Golgi GFP BacMan 2.0 (C10592), lysosome-RFP (C10597), mitochondria-GFP (C10600), actin-RFP (C10583), or endoplasmic reticulum-GFP BacMan (C10590) (Invitrogen, Eugene Oregon). Prolong Gold, containing 10 μg/ml DAPI (P36935), was used to mount the slides and stain the nuclei. After the mounting medium dried, slides were sealed and then observed with a combination of dark field and fluorescence microscopy.

Primary hippocampal neurons were transfected with Mitochondria-GFP (Invitrogen) at DIV14, and fixed in 10% buffered formalin at DIV 20–22. Then, samples were incubated with anti-Flag (1:500 dilution, Origene, Rockville, MD) overnight at 4 °C. Cells were washed and incubated with Alexa-568 antibody (1:200 dilution, Invitrogen, Molecular Probes, Carlsbad, CA) for 1 h at room temperature and mounted on glass slides. Images were taken with Zeiss LSM 710 confocal scanning microscope (Zeiss, OberKDchen, Germany) and processed using ZEN software (Carl Zeiss Microscopy GmbH, Jena, Germany).