Leica sp5 inverted confocal microscope

Samples were fixed in 4% paraformaldehyde at 4°C for 1 h and immunofluorescently analysed as described previously (Niakan and Eggan, 2013 (link)). The primary antibodies (all at 1:500 dilution) used include: anti-Oct4 (sc-5279, sc-8628 or sc-9081, Santa Cruz Biotech), anti-Nanog (AF1997 R&D, REC-RCAB0001P 2B Scientific, or ab21624, Abcam), anti-Cdx2 (MU392A-UC, Biogenex), anti-Klf17 (HPA024629, Atlas), anti-Ap2γ (AF5059, R&D), anti-Sox17 (AF1924, R&D) and anti-Foxa2 (3143, Cell Signaling). Embryos were imaged on a Leica SP5 inverted confocal microscope (Leica Microsystems).

Pegs designated for confocal microscopy were snapped off the lid using sterile tweezers and stained with a SYTO 9/propidium iodide (LIVE/DEAD, BacLight; Invitrogen, Waltham, MA, USA) solution. The pegs were incubated, covered from the light, for 20 min. After incubation, the pegs were rinsed in PBS and placed on 50 mm glass-bottom dishes (MatTek, Ashland, MA, USA). The pegs were imaged using a Leica SP5 Inverted Confocal Microscope (Leica Microsystems, Buffalo Grove, IL, USA) at a resolution of 512 × 512 pixels using a 63× water immersion objective (63×/1.2W).

The cells were fixed with methanol for 10 min. The cytoplasm was stained with neutral red (pink) and then stained with Prussian blue (blue) to highlight the iron-laden particles. Light microscopy images were generated at 1, 3, 6 and 24 h post-exposure to 5, 10 and 25 µg/mL of FeCaP NPs. Confocal fluorescence microscopy was also used to establish intracellular FeCaP NP localization using 3D image analysis of TT1 cells. TT1 cells were grown to high confluence and exposed to 25 μg/mL FeCaP NPs in serum-free RPMI-1640 medium. Cells were washed to remove apical FeCaP excess then fixed with 4% paraformaldehyde. Samples were then incubated for 15 min at room temperature with wheat germ agglutinin (WGA) stain conjugated to Alexa Fluor 647 (1:100; Invitrogen™, Waltham, MA, USA) to reveal the cell membrane. Cells were washed in PBS and then counter-stained with DAPI (1:100; Invitrogen™, Waltham, MA, USA) for 15 min at RT and rinsed 3 times with PBS before imaging. FeCaP particles were revealed using a 488 nm laser and PMT2 483–496 nm for reflection. Z-Stacks of 3 different samples were taken using the Leica SP5 inverted Confocal Microscope (Leica Microsystems, Wetzlar, Germany) with an optical zoom of 63×. The Z-Stacks depth was chosen in order to include the entire height of the TT1 cells monolayer. Image analysis was performed using the FIJI image analysis software.