Fv10 asw 4.2 viewer image software

For immunofluorescence staining of tissues, 10 μm cryosections were equilibrated at RT, fixed in PFA for 15 min, permeabilized using PBS containing 0.2% Triton-X for 10 min, and blocked in PBS containing 0.05% Tween-20, 5% FBS, 5% BSA, and 5% goat serum (GE Healthcare, UK) for 1 h. The sections were immunostained with rabbit anti-fluorescein (cat. no. A889, Thermo Fisher Scientific, MA, USA), rat anti-mouse CD31, biotin rat anti-mouse CD11b (cat. no. 553370; 557395, BD Biosciences, CA, USA), rat anti-mouse LYVE-1 (cat. no. 14044382, eBioscience, CA, USA), rabbit polyclonal anti-Ki67 (cat. no. NB500–170, Novusbio, UK), and rabbit anti-Cleaved Caspase-3 (Asp 175),(cat. no. 966, Cell Signaling Technology, Inc., MA, USA) as primary antibodies,. Alexa 488-conjugated goat anti-rabbit IgG, Alexa 647-conjugated goat anti-rat IgG, Alexa 546-conjugated goat anti-rabbit IgG (all Invitrogen, Thermo Fisher Scientific, MA, USA) and strepta- vidin Dylight^® 550 (Thermo Fisher Scientific, MA, USA) were used as secondary antibodies. Nuclei were counterstained with 10 pg/ml DAPI. The stained tissue sections were examined by fluorescence confocal microscopy using Olympus FV1200MPE instrument, and the images were processed and analyzed using the FV10-ASW 4.2 Viewer image software (Olympus, Germany) and Image J freeware.

For flow cytometry MKN-45P, CT-26, SKOV-3 cells in suspension were incubated with NWs in complete cell culture medium for 1 h The NW-containing solution was removed, cells were washed and analyzed by flow cytometry (Accuri, BD Biosciences, CA, USA). Anti-p32 antibody inhibition was done by pre-incubating the cells in suspension with 20 μg/ml of in-house rabbit polyclonal p32 antibody, followed by NW incubation for 1 h, washes and flow cytometry.
For confocal imaging of FAM-labeled NWs, MKN-45P, CT-26, SKOV- 3 (5 × 10⁴ cells) were seeded on glass coverslips in a 24-well plate. After 24 h, NW samples at 40 μg Fe/well were added to the cells and incubated at 37 °C for 3 h. The cells were washed with PBS, fixed with 4% of paraformaldehyde in PBS pH 7.4 (PFA), co-stained with DAPI, and imaged with fluorescence confocal microscopy (Olympus FV1200MPE, Germany).
For immunofluorescence staining, the cultured cells were incubated with rabbit anti-fluorescein (cat. no. A889, Thermo Fisher Scientific, MA, USA) primary antibody to detect the NWs and with mouse anti- cytochrome-C (cat. no 89918, Thermo Fisher Scientific, MA, USA) to label mitochondria. The images were analyzed using the FV10-ASW 4.2 Viewer image software (Olympus, Germany). Optionally, iron was stained using the Prussian Blue cytochemistry [33 (link)] followed by coun- terstaining with Nuclear Fast Red.