Goat anti rabbit igg tr

To analyze whether abortigenic and neurovirulent EHV1-infected T lymphocytes express viral proteins on the cell surface, double immunofluorescence staining was performed. At 9 hpi, T lymphocytes were fixed in 1% PFA, followed by cytospin centrifugation. The nonspecific binding sites (e.g., equine IgG receptor) were first blocked by incubation with 10% negative horse serum, obtained during a previous in vivo study by Vairo et al. (96 (link)), for 45 min at 37°C. Next, T lymphocytes were incubated with avidin and biotin (Thermo Fisher Scientific) to reduce nonspecific binding for 15 min at 37°C. To label viral proteins on the cell surface, a biotinylated horse anti-EHV1 pAb (1:20) (81 ) was used for 1 h at 37°C. Next, cells were permeabilized with 0.1% Triton X-100 for 2 min at RT, followed by incubation with a rabbit pAb against IEP (1:1,000) to visualize IEP expression. Subsequently, cells were incubated for 50 min at 37°C with streptavidin-FITC (1:100) and goat anti-rabbit IgG–TR (1:100) (Molecular Probes). Nuclei were counterstained with Hoechst 33342 dye (10 μg ml⁻¹).

To determine viral transmission from EHV1-inoculated RK13 cells or EREC to T lymphocytes, double immunofluorescence staining was carried out. RK13 cells and EREC with potential bound T lymphocytes were incubated for 1 h at 37°C with a rabbit anti-IEP pAb (1:1,000) to visualize IEP and a mouse MAb against CD3 (IgG1; 1:50) to visualize T lymphocytes. Subsequently, cells were incubated for 50 min at 37°C with goat anti-rabbit IgG–TR (1:100) and goat anti-mouse IgG1–FITC (1:100) (Molecular Probes). Nuclei were counterstained with Hoechst 33342 dye (10 μg ml⁻¹).
Triple immunofluorescence staining was performed to visualize viral reactivation in the EHV1-inoculated monocytic cells and viral transfer between EHV1-inoculated monocytic cells and T lymphocytes. Adherent cells and cytospin-centrifuged nonadherent cells were incubated with a rabbit pAb against IEP (1:1,000) to visualize IEP, a biotinylated pAb against EHV1 (1:20) (8 (link)) to visualize late viral proteins, and a mouse MAb against CD3 (IgG1; 1:50) to stain T lymphocytes. Next, cells were incubated for 50 min at 37°C with goat anti-rabbit–TR (1:100), streptavidin-FITC (1:200), and goat anti-mouse IgG1–Alexa Fluor 647 (1:100) (Molecular Probes). Nuclei were counterstained with Hoechst 33342 dye (10 μg ml⁻¹).