Axiovision fluorescence microscope

Term placental explants were transfected with NT or p32-specific siRNA sequences and cultured for 72 h, then were subjected to a pulse-chase experiment. Explants were incubated with MitoTracker Red CMXRos (10 nM; Life Technologies) for 6 h to allow passive transfer into the syncytium, then were transferred to fresh culture medium for a further 18 h to allow accumulation of dye in the mitochondria to occur (i.e. up to 96 h post-transfection). MitoTracker Red CMXRos is a mitochondrial membrane potential-sensitive, red fluorochrome that accumulates in active mitochondria (Pendergrass et al., 2004 (link)). Explants were washed in PBS, fixed in neutral buffered formalin [4% (v/v); 24 h] and embedded in Optimal Cutting Temperature cryopreservation medium (RA Lamb; UK). Tissue sections (10 µm) were fixed in ice-cold methanol, washed twice in PBS (2 × 5 min), mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, UK) and visualized using a Zeiss Axiovision fluorescence microscope. Images were captured at the same exposure so that comparisons across samples could be made.

TUNEL staining on rat β-cells was performed 48 h after transfection using the TMR red In Situ Cell Death Detection Kit (Roche) combined to polyclonal guinea pig anti-insulin (dilution 1:40, PA1-26938 Invitrogen) followed by incubation with goat anti-guinea-pig AlexaFluor 488 antibody (dilution 1:400, A11073 Thermofisher). Cell nuclei were stained with Hoechst 33342 (1 μg/ml, Invitrogen). Coverslips were mounted on microscope glass slides with Fluor-Save mounting medium (VWR International SA) and were visualized with a Zeiss Axiovision fluorescence microscope. A minimum of 10³ cells were counted per condition. Incubation for 24 h with a mix pro-inflammatory cytokines (1 ng/mL IL-1β, 10 ng/mL TNF-α and 30 ng/mL IFN-γ) was used as positive control. Experiments were performed in single replicates.

Imaging was performed using a Zeiss AxioVision fluorescence microscope and Zeiss Axiovision software, Zeiss LSM-510 Meta, LSM-800 and LSM 880 confocal microscopes and Zeiss Zen Blue acquisition software, or using a Nikon A1 Confocal Laser Microscope System NIS-Elements software. Images were processed with Imaris and Adobe Photoshop software. Imaris colocalization function was used to produce pictures showing p-MLC2 (Figure 4b), vinculin (Figure 4c) or paxillin (Figure 4—figure supplement 1b) staining that is associated with F-actin staining.

Animals were killed and the pancreases fixed in 10% neutral buffered formalin (Sigma-Aldrich) for 24 h and embedded into paraffin for sectioning. The slices were stained using the following primary antibodies: 1:500 rabbit anti-Ki67 (Abcam: # ab15580), 1:200 guinea pig anti-insulin (Abcam: # ab7842), 1:200 rabbit anti-glucagon (Abcam: # ab10988). Goat anti-rabbit Alexa-Fluor-488, goat anti-guinea pig Alexa-Fluor-555 and goat anti-mouse Alexa-Fluor-555 diluted at 1:400 (Invitrogen: # A11008, # A21435 and # A21422, respectively) were used as secondary antibodies. TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche). Cell nuclei were visualized with Hoechst 33342. Images were collected using a Zeiss Axiovision fluorescence microscope.

The cellular uptake of doxorubicin delivered with Au@PEG-INU NPs was evaluated by fluorescence microscopy analysis. Cells were added to 8-well glass bottomed slides at a concentration of 2 × 10⁴ per well and allowed to adhere overnight. Au@PEG-INU/Doxo and free doxorubicin were added at a final concentration of 40 μg mL^–1 of Au⁰, corresponding to ∼4 μg mL^–1 doxorubicin, and cells were incubated for either 4 h, 24 h or 48 h. After each incubation time, cells were washed using PBS and fixed using 4% formaldehyde at room temperature for 20 minutes followed by washing with PBS. Cells were stained first for the nucleus using 4′,6-diamidino-2-phenylindole (DAPI) diluted 1/600 in cell media, and then for the membrane using wheat germ agglutinin-Alexa Fluor® 647 conjugate (WGA647) (1/200 in PBS). Both incubations were carried out at room temperature for 20 min followed by extensive washing. Cells were viewed using a Zeiss AxioVision fluorescence microscope with transmitted light and filters for DAPI, plasma membrane and doxorubicin. To study the tumor selectivity of Au@PEG-INU/Doxo, A549, MDA.MB.435s or MCF-7 cells were co-cultured with normal epithelial cells and/or fibroblasts in glass bottomed 96-well plates (Ibidi; 1.5 × 10⁴ per well) and the passive targeting and uptake of doxorubicin and Au@PEG INU/Doxo were imaged (cf. SI 3.2, ESI†).