Appendix 4 of Faralli, Mol Vis 2019; 25:237-254.

Appendix 4. Immunofluorescence microscopy of FLAG tagged-α2-chimaerin expression.

To access the data, click or select the words “Appendix 4.” FLAG tagged-α2-chimaerin (arrows) was only detected along the edges of the cell membranes in TM-1 cells transfected with the pcDNA3.1-CHN1-DYKDDDK plasmid and not in the mock transfected cells (no plasmid added). In contrast, only diffuse staining was detected in cultures labeled with IgG control. Scale bar=100µm. Forty-eight hrs post-transfection, TM-1 cultures were processed for immunolabeling as we previously described (22). Cultures were fixed with 4% paraformaldehyde and permeablized with 0.2% Triton X-100 in PBS. Fixed cells were blocked with 1% BSA and then stained with a 1/100 dilution of either a IgG isotype control or anti-FLAG IgG (F1804, Sigma) for 1 hr. Primary antibodies were detected with Alexa 488-conjugated goat anti-mouse IgG diluted 1/500 for 30 min.