Immunocytochemistry
To visualize the cellular localization of EFEMP1 protein, cells
were seeded on clear coverslips (Neuvitro Corp., Camas, WA). In order to
visualize the endoplasmic reticulum, CellLight™ ER-RFP, BacMam 2.0
(Invitrogen, Carlsbad, CA) was added to each well during transfection.
The cells were processed for immunocytochemistry 48 hours after
transfection. Condition media was aspirated, coverslips washed with PBS,
and fixed in 4% PFA. Cells were permeabilized using 0.5% Triton X-100
and blocked in 3% BSA with PBS, followed by incubation in V5 Tag
Monoclonal Antibody (Thermofisher, Waltham, MA) overnight. Coverslips
were retrieved and incubated in Goat anti-Mouse IgG (H+L) Highly
Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen,
Carlsbad, CA) for 2 hours and DAPI (Thermofisher, Waltham, MA) for 1
minute. Coverslips were mounted on clear slides with ProLong™ Glass
Antifade Mountant (Invitrogen, Carlsbad, CA). Imaging of the transfected
cells was done using a confocal laser scanning microscope (SP8, Leica
Microsystems, Buffalo Grove, IL).