Triton x 100

For subcellular localization experiments, a dually labeled assembly
was used with ASO labeled with Cy5.5 at the 5′ end and rHA
labeled with Cy3. C28/I2 cells were seeded overnight at 10,000 cells/well
in an 8-well chamber slide (Sarstedt, cat# 94.6140.802) at 37 °C,
5% CO₂. The cells were then treated with the dually labeled
assembly at 100 nM and incubated for further 4, 6, and 24 h at 37
°C, 5% CO₂. Cells were washed with DPBS and then fixed
with formalin 10% (Sigma-Aldrich, cat# HT5014) for 20 min. After further
washing, cells were permeabilized with 0.1% Triton X-100 (Cell Biolabs,
INC., cat# 124102) for 20 min, followed by blocking with 5% bovine
serum albumin (BSA, Sigma-Aldrich, cat# A9647) in DPBS for 1 h. Cells
were washed and incubated with primary rabbit polyclonal antibodies
(Abcam), (anti-EEA1, cat# ab2900, for early endosomes, and anti-Lamp1,
cat# ab24170, for lysosomes) in 5% BSA at 4 °C overnight. The
cells were then washed and incubated with goat antirabbit secondary
antibody, Alexa Fluor 488 (Invitrogen, cat# A11034) for 90 min, followed
by washing and mounting the slide with UltraCruz Aqueous Mounting
Medium with DAPI (Santa Cruz Biotechnology, Inc., cat# sc-24941).
The slides were kept at 4 °C in the dark until visualization
on a Zeiss Confocal microscope LSM 700 (Carl Zeiss MicroImaging).
Images were processed with Zeiss Zen Black 2012 edition.