Fluoview fv1200

Intracellular
iron content was investigated by using the fluorescent probe Phen
Green SK (PGSK; Invitrogen) and FerroOrange (Dojindo Laboratories)
according to the manufacturer’s instructions.. SKOV3 and OVCAR3
cancer cells were treated (control, olaparib, or olaparib-Ga) for
3 h, then incubated with 5 mM PGSK or 0.5 μg/mL FerroOrange
at 37 °C for 30 min. For PGSK, the cells were then washed with
PBS twice (washing was not necessary after FerroOrange treatment).
Fluorescence images were recorded via a confocal
laser scanning microscope (Olympus, Fluoview FV1200). The experiments
were repeated independently three times.
Intracellular ROS generation
was measured using a confocal laser scanning microscope (CLSM). SKOV3
and OVCAR3 cancer cells were cultured in CLSM culture dishes (2 ×
10⁵ cells per well) overnight under normal culture conditions.
The culture medium was then replaced with an equal volume of DMEM
containing control, olaparib, or olaparib-Ga and incubated for 3 h.
PBS containing 2.5 μM ROS was used to replace the medium, and
the cells were then incubated at 37 °C for 30 min in the dark.
The cells were then washed three times. Green fluorescence was detected
to confirm intracellular ROS generation using a confocal laser scanning
microscope (Olympus, Fluoview FV1200). The experiments were performed
independently three times.

Mice were euthanized at P17 and eyes were enucleated and fixed with freshly prepared 4% paraformaldehyde (PFA) for 1-2 h at room temperature (RT). Corneas were removed with scissors along the limbus and the whole retinas were dissected. Then, they were blocked and permeabilized in Tris-buffered saline (TBS) containing 5% Bovine Serum Albumin (BSA) and 0.1% Triton-X-100 during 6 h at 4°C. After that, retinas were incubated ON with 0.02 μg/μL of Isolectin IB4 Alexa fluor-488 conjugate (GSA-IB4) from Molecular Probes, Inc. (Eugene, OR, USA). Retinas were then washed with TBS containing 0.1% Triton-X-100, stored in PBS at 4°C and examined by confocal laser-scanning microscopy (Olympus FluoView FV1200; Olympus Corp., New York, NY, USA).

Adult thoracic muscle was dissected and then fixed in 4% paraformaldehyde in PBS. The tissue was blocked for one hour in PBS with 0.1% Triton X-100 and 10% fetal bovine serum, then incubated overnight in 1∶500 rabbit anti-GFP (#A11122, Life Technologies) and either 1∶500 mouse anti-Myc 9E10, 1∶500 mouse anti-FLAG (#F3165, Sigma), or 1∶2000 mouse anti-Cytochrome C (Cyto C) (#556433, BD Biosciences). The tissue was then washed in PBS with 0.1% Triton X-100 and incubated overnight with 1∶500 anti-rabbit Alexa 488 secondary antiserum (#A11034, Life Technologies) and 1∶500 (1∶1000 for Cyto C) anti-mouse Alexa 568 secondary antiserum (#A11031, Life Technologies). After final washes, the tissue was mounted in Prolong Gold (#P36934, Invitrogen) and imaged sequentially with 488 nm and 561 nm lasers on an Olympus FluoView FV1200 (Olympus America) with a 60× oil objective and 15× digital zoom, taken at 1024×1024 pixels and 30 steps of 0.13 µm.