Immunocytochemistry

To assess endosomal internalization, we incubated 100 pmol Cy3-conjugated A01B aptamer with C2C12 myoblasts for 30–120 min at 37°C as described earlier under the section Confocal Microscopy. Following fixation, cells were processed for immunocytochemical analysis of the EEA1 early endosomal marker. To remove excess fixative, we washed cells twice for 5 min with 1 mL of ice-cold PBS and then permeabilized them with 3 mL of ice-cold 100% methanol for 3 min in the freezer. Cells were then washed 3× with 1 mL of PBS supplemented with Tween 20 (Scharlau, Barcelona, Spain) at a final concentration of 0.1% (PBS-T). Blocking of non-specific binding was performed with 5% normal goat serum (Biosera, Nuaillé, France) in PBS-T for 60 min at room temperature. Coverslips were transferred in a humidity chamber; then the primary antibody rabbit EEA1 (C45B10) (Cell Signaling Technologies, Danvers, MA, USA) was applied at 1:100 in blocking buffer and incubated overnight at 4°C. The next day, coverslips were washed 3× with 1 mL of PBS-T and then incubated with Invitrogen Alexa Fluor Plus 488 goat anti-rabbit IgG secondary antibody at a dilution of 1:500 in blocking buffer for 60 min at room temperature. Coverslips were washed 3× 5 min with 1 mL of PBS-T; then TO-PRO-3 nuclear stain was applied for 15 min at room temperature at a dilution of 1:1,000 in PBS. Coverslips were washed 2× with 1 mL of PBS and mounted with Dako fluorescent mounting medium onto Poly-Prep slides. Slides were left to dry for 30–60 min at room temperature. Images were taken on a Leica TCS SP5 confocal microscope. Single-cell magnifications were achieved by applying the 60× oil objective followed by additional electronic magnification.