Goat anti rabbit alexa fluo488

The three cell lines were transfected the miR-224-mimics/inhibitor as described above. Cells were fixed in 4% paraformaldehyde and permeabilized by 1% Triton X-100. Following blocking with 1% bovine serum albumin, cells were serially incubated in rabbit anti-LC3 (Abcam) and Goat anti-rabbit Alexa Fluo488 (Invitrogen). The images were acquired using the AV300-ASW confocal microscope (Olympus America Inc., Center Valley, USA) with a 60 × oil lens. The pictures analysed using Image-Pro Plus 6.0 (Media Cybernetics)32 (link).

Cells grown on cover glasses were treated as designated. Cells were fixed in 4% paraformaldehyde and permeabilized by 0.5% Triton X-100. Following blocking with 3% bovine serum albumin, cells were serially incubated in rabbit anti-LC3B (Sigma-Aldrich) and Goat anti-rabbit Alexa Fluo488 (Invitrogen). Finally, cells were rinsed and mounted on cover glasses with Prolong Gold anti-fade reagent with 4′-6-diamidino-2-phenylindole (Invitrogen) and the immunostaining was observed under the AV300-ASW confocal microscope (Olympus America Inc., Center Valley, USA) with a 60 × oil lens. The number of LC3 puncta per cell was quantified manually. At least 20 cells in 3 independent experiments were analyzed randomly.

Cell grown on cover glasses were treated with various concentration of SPIONs with or without SMF as designed. After 24 hours, the cells were rinsed with PBS, fixed in 4% paraformaldehyde and permeabilized by 03% Triton X-100. After blocking in 3% bovine serum albumin, the cells were serially incubated in rabbit anti-LC3B (3868S, Cell Signaling Technology, Inc. USA, 1:200) at 4°C overnight and Goat anti-rabbit Alexa Fluo488 (Invitrogen, 1:3000) for 1 hour at room temperature in the dark. Then the cells were stained with nuclear stain DAPI (10 μg/mL) for 5 minutes at room temperature and the image was observed under a confocal microscope (FV 1000; Olympus Corporation, Tokyo, Japan).