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).