Mouse igg1 kappa isotype control

Fluorescence microscopy analysis
was used to show the colocalization of CD71 and HFn-FITC within CD71-positive
C666-1 cells. Upon reaching 90% confluence, C666-1 cells were detached,
washed, and then fixed with 4% paraformaldehyde for 15 min at 37 °C.
After washing and suspending in phosphate-buffered saline (PBS), a
20 μL aliquot of the cell suspension was placed onto a slide,
allowed to sit for 20 min at room temperature, and then dried in an
oven at 37 °C. The cell smears were permeabilized with 0.1% Triton
X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for
1 h at room temperature. The cells were then incubated with the following
reagents: 5 μg/mL of mouse IgG1 kappa isotype control (Invitrogen,
USA), 5 μg/mL of CD71 mouse monoclonal antibody (Invitrogen,
USA), and 10 μg/mL of HFn-FITC in 0.1% BSA at 4 °C overnight.
Subsequently, the cells were labeled with Alexa Fluor 488 and Alexa
Fluor 555 secondary goat antimouse antibody (Bioss, China) at a dilution
of 1:100 for 1 h at room temperature. After rinsing with PBS containing
0.05% tween 20 (Solarbio, China), an antifade mounting medium with
DAPI (Beyotime Biotechnology, China) was applied to stain the cell
nuclei for 10 min. Finally, the cell smears were examined using a
confocal laser scanning microscope with 405, 488, and 543 nm wavelengths
of laser (Zeiss LSM880, Carl Zeiss AG, Oberkochen, Germany).