Eclipse te300 fluorescent microscope

To investigate cellular uptake, live-cell fluorescence microscopy was used to image GIN8, GIN28 and U87 cells. NPs for uptake experiments were prepared as previously described but were formulated as 10% wt/wt of mPEG₅₀₀₀-(LA)₅₀-(TBPC)₅₀-Cy5 (P2-Cy5) and 90% wt/wt mPEG₅₀₀₀-(LA)₅₀-(TBPC)₅₀ (P2) and diluted to a final concentration of 50 µg/mL in 10% (v/v) FBS containing DMEM (no phenol red). GIN8, GIN28 and U87 cells were seeded in CellView™ 35 mm diameter glass-bottom cell culture dishes at a density of 2.5 × 10⁵ cells per dish and cultured for 24 h. Cy5-NPs (50 µg/mL) were incubated with cells for 0.5, 2 and 4 h at 37 °C with 5% CO2. Following exposure, NP solutions were removed and cells washed three times with ice-cold PBS. Cells were then stained with 10 µg/mL Hoechst 33342 (Thermo-Fisher) or 10 µg/mL Hoechst 33342 and 50 nM Lysotracker green DND-26 (Thermo-Fisher) applied in Hank’s Balanced Salt Solution (HBSS) for 30 min. Staining solution was removed and cells washed twice with PBS. FluoroBrite DMEM was added to wells and cells were imaged on an inverted Nikon Eclipse TE 300 fluorescent microscope on DAPI, FITC and Cy5 filters. Images were processed using ImageJ software (1.52f) and the Coloc 2 plug in was used for calculation of Pearson’s correlation coefficient for co-localisation studies.

Cells were cultured in 8-well Lab-Tek Chamber Slides (Cat. no. 177402) for indicated time period. Cells were fixed, permeabilized, blocked, and stained as described previously^{28 (link)}. For indirect immunofluorescent cell staining, an anti-FLAG M2 monoclonal antibody and an Alexa Fluor 594-conjugated goat anti-mouse antibody were used to detect FLAG-CASZ1b or mutant constructs. An anti-MHC antibody and an Alexa Fluor 594-conjugated goat anti-mouse antibody were used to detect MHC. Stained cells were imaged and analyzed using a Nikon Eclipse TE300 fluorescent microscope.

Cells were seeded onto microscope slides and fixed with 4% paraformaldehyde. Slides were blocked with 5% BSA (Sigma) + 1X TBST at room temperature for 1 h and then probed overnight at 4°C with anti-HIF-1α antibody (1:100 dilution). Slides were washed and probed with a Rhodamine Red Goat anti-mouse secondary antibody (Molecular Probes/Invitrogen) at a 1:200 dilution in 5% BSA. Localization and expression was visualized with a Nikon Eclipse TE-300 fluorescent microscope with excitation at 518 nm and emission at 605 nm. Fluorescent images were captured using a CoolSnap digital camera with Metamorph imaging software (Molecular Devices, Sunnyvale, CA).