Af488 conjugated goat anti mouse igg2b

ARPE-19 cells on glass coverslips were inoculated with freshly harvested cell-free VZV EMC-1 edited by sgRNA8-2 and incubated for 3 days at 37 °C. Infected cells were fixed with 4% paraformaldehyde (PFA), permeabilized for 10 min with 0.1% Triton X-100 in PBS, blocked with 5% goat serum diluted in PBS + 0.05% Tween and incubated with monoclonal mouse IgG1 anti-ORF8 antibody (1:100, generously provided by Dr. S. Jonjic, University of Rijeka, Rijeka, Croatia [16 (link)]) and monoclonal mouse IgG2b anti-VZV glycoprotein E antibody (MAB8612, Millipore, Amsterdam, The Netherlands) diluted PBS + 0.05% Tween 20 (PBS-T) overnight at 4 °C. Cells were washed with PBS-T and incubated with AF488-conjugated goat anti-mouse IgG2b and AF555-conjugated goat anti-mouse IgG1 antibodies (Thermo Fisher Scientific, Breda, The Netherlands) diluted 1:500 in PBS-T. Finally, the coverslips were washed, incubated for 5 min in PBS with Hoechst 33342 (1:1000, Life Technologies, 20 mM), washed in PBS and mounted using Prolong Gold Antifade Mounting medium (Thermo Fisher Scientific, Breda, The Netherlands). Stained cells were analyzed using a Zeiss LSM 700 confocal laser scanning microscope (Zeiss, Oberkochen, Germany) with a magnification of 100× or 400×. Photoshop CC 2021 software (Adobe, San Jose, CA, USA) was used to adjust brightness and contrast.

Paraffin embedded sections were visualized using FITC channel only by the Zeiss LSM800 confocal microscope. For evaluating phagocytosis, SF samples were initially collected as described above. SF cells were filtered and stained with PE-anti-Ly-6G (1A8, BioLegend) plus Hoechst (33342, Invitrogen, ThermoFisher Scientific) for 30 min at ambient temperature. After the staining, cells were fixed, permeabilized (BD Cytofix/CytoPerm solution, described above), and further stained intracellularly with AF488-conjugated goat anti-mouse IgG2b (polyclonal, ThermoFisher Scientific) in 1X Perm solution for 1 h. After sufficient wash by the same Perm solution, cells were finally visualized under the 100X objective of the Zeiss LSM800 system using AF488, PE, and Hoechst channels, with compensations been optimized.

Paraffin embedded sections were visualized using FITC channel only by the Zeiss LSM800 confocal microscope. For evaluating phagocytosis, SF samples were initially collected as described above. SF cells were filtered and stained with PE-anti-Ly-6G (1A8, BioLegend) plus Hoechst (33342, Invitrogen, ThermoFisher Scientific) for 30 min at ambient temperature. After the staining, cells were fixed, permeabilized (BD Cytofix/CytoPerm solution, described above), and further stained intracellularly with AF488-conjugated goat anti-mouse IgG2b (polyclonal, ThermoFisher Scientific) in 1X Perm solution for 1 h. After sufficient wash by the same Perm solution, cells were finally visualized under the 100X objective of the Zeiss LSM800 system using AF488, PE, and Hoechst channels, with compensations been optimized.