After GA-osmium fixation of fibroblast I experienced ghost mitochondria in TEM. Have you experienced that the rest of the cell is fixed properly but not for mitochondria?
Protocol
Fix solution: 2,5% GA, 2% FA, PHEM pH adj. to 7.4 with 0.2M NaOH
Fixation: 10min at RT (with RT-tempered fix solution), then on ice for 1h
Wash: 3 x 5min with PHEM pH=7.4, on ice
Osmication: 1% OsO4 in miliQ, 1h in the fridge
Wash: 3 x 5min with PHEM pH=7.4, on ice
Dehydration: cold EtOH series 30-50-70-90-100% 3-5min on ice; Anhydrid EtOH 2 x 10min on ice
Thank you for any help.
Lenka