Goat anti human eea 1 antibody

Cells were plated on coverslips (BD Biosciences) (10⁴ cells/coverslip) and cultured in their respective complete growth media. After treatment with HApt-AuNS, the coverslips were washed with PBS twice to remove all residual growth media. Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X100 for 5 min. The coverslips containing fixed cells were then blocked with solution containing 1% BSA and 5% normal donkey serum for 1 h at room temperature. The cells were then incubated at 4 °C overnight with primary antibodies (1:200) in blocking solution (mouse anti human LAMP-1 antibody (Santa Cruz) and goat anti human EEA-1 antibody (Santa Cruz)). After overnight incubation, the coverslips were washed 6 times with PBS for a total of 1 h. Secondary antibodies in blocking solution were incubated with the coverslips for 1 h at room temperature (donkey anti goat IgG Alexa 488 and donkey anti mouse IgG Alexa 568). The coverslips were washed for 1 h with PBS, and cell nuclei were counterstained with DAPI. The coverslips were then mounted on glass slides using gold-antifade mounting media (Invitrogen).

After plating on the coverslips, cells were treated with Cy5-labeled 13-nm, 50-nm or AuNS nanoconstructs which had the similar density of RNA strands (0.21, 0.19 and 0.18 molecules nm⁻² for 13-nm spheres, 50-nm spheres and 40-nm stars, respectively). After 2 h or 24 h incubation, the coverslips were washed 3 times with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X100. Cells were incubated with the blocking solution (1% BSA and 10% rabbit serum in PBS) at room temperature for 1 h. Cells were then incubated at 4 °C overnight with a solution of primary antibodies (mouse anti-human LAMP-1 antibody and goat anti-human EEA-1 antibody; 300-fold dilutions of stocks from Santa Cruz Biotechnology) in blocking solution. After overnight incubation, coverslips were washed 6 times with PBST (0.1% Tween 20 in PBS) over 1 h. Secondary antibodies (rabbit anti-mouse IgG (H + L) Alexa Fluor 488 and rabbit antigoat IgG (H + L) Alexa 594, Thermo) in blocking solution were incubated with the cells for 1 h at room temperature. Following an additional 6 washes with PBST buffer over 1 h, coverslips were mounted on glass slides using DAPI-containing antifade mounting medium (Invitrogen).