Prolong gold antifade solution

HFFs were grown on glass coverslips within 24-well plates and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) (37 (link)). Cells were permeabilized with 0.1% Triton X-100 in PBS prior to blocking with 5% normal goat serum in PBS. Coverslips were then incubated with monoclonal antibodies or polyclonal primary antibodies in 5% normal goat serum in PBS with 0.2% Tween 20 (PBST). When using rabbit polyclonal antibodies, the ChromPure human IgG Fc fragment (Jackson ImmunoResearch, West Grove, PA) was included for all primary and secondary antibody incubations. After incubation with primary antibodies, coverslips were washed in PBS containing 0.1% Tween 20. Alexa Fluor secondary antibodies were used in this study, including Alexa Fluor 488 (green), Alexa Fluor 594 (red), and Alexa Fluor 647 (gray) (Life Technologies, Carlsbad, CA). Nuclei were identified by 4′,6-diamidino-2-phenylindole (DAPI) staining. Coverslips were mounted with ProLong gold antifade solution (Cell Signaling Technology). The images were acquired using Olympus FV1000 confocal microscopy systems and processed using FluoView software.

HF cells were grown on glass coverslips within 24-well plates and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Cells were permeabilized with 0.1% Triton X-100 in PBS prior to blocking with 5% normal goat serum in PBS. Coverslips were then incubated with monoclonal antibodies or polyclonal primary antibodies in PBS containing 10% normal goat serum. When using rabbit polyclonal antibodies, the ChromPure human IgG Fc fragment (Jackson ImmunoResearch, West Grove, PA) was included with all primary and secondary antibody incubations. After incubation with primary antibodies, coverslips were washed in PBS containing 0.1% Tween 20. Alexa Fluor secondary antibodies were used in this study, including Alexa Fluor 488 (green), Alexa Fluor 594 (red), and Alexa Fluor 647 (blue) (Life Technologies, Carlsbad, CA). Nuclei were identified with 4′,6-diamidino-2-phenylindole (DAPI) staining. Coverslips were mounted with ProLong gold antifade solution (Cell Signaling Technology, Danvers, MA). The Golgi membrane length was measured using the Trace feature in FluoView software (Olympus Corporation), and the results are detailed in Fig. S1 in the supplemental material.