Chrompure human igg fc fragment

Cell monolayers were washed with phosphate buffered saline (PBS) and cells were either fixed with 4% paraformaldehyde in PBS or methanol and were permeabilized using 0.2% Triton X-100 in PBS. When specified, cells were first permeabilized using buffer (0.1% Triton, 100 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 1 mM Hepes) in order to deplete soluble proteins from the cytosol, and subsequently fixed with 4% paraformaldehyde in PBS. Cells were then incubated for 1 h in PBS, gelatin 0.2% supplemented with 5% of goat serum for blocking, and then with appropriate primary antibodies. The cells were washed 3 times in PBS Tween20 0.1%, and then incubated with appropriate secondary antibodies. When using rabbit polyclonal antibodies, ChromPure human IgG Fc fragment (Jackson ImmunoResearch) was added in blocking buffer, as well as with primary and secondary antibody incubations, in order to limit cross-reaction with Fc receptor-like HCMV proteins³⁵ . Coverslips were mounted in Glycergel (Dako, C0563) and examined using a Zeiss Axiovert 200 M epifluorescence microscope (Zeiss instruments) or a Leica TCS SP8 confocal microscope (IPSIT, cellular imaging, Châtenay-Malabry, France). Photographic images were resized, organized, and labeled using ImageJ software or LAS AF Lite.

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.