Alexa fluor 555 conjugated anti mouse igg

PK-15 cells were infected with JS-2020 at 0.1 MOI in DMEM and supplemented with 1% FBS; cell samples were collected at 24 h post infection. For immunofluorescence, the infected PK-15 cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 at room temperature. Cells were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature and incubated with PRV gB protein antibody (A general gift from Prof. Beibei Chu at Henan Agricultura University) overnight at 4 °C. Following three washes with PBS, cells were incubated with an Alexa fluor 555-conjugated anti-mouse IgG (Cell Signaling Technology, Danvers, MA, USA) for 1 h at the room temperature. The cells were visualized with an inverted fluorescence microscope (U-HGLGPS, OLYMPUS, Tokyo, Japan).

The Ana2014 cells were washed twice with PBS and allowed to attach on poly-lysine-coated coverslips. The cells were fixed and permeabilized in 3.7% paraformaldehyde/0.18% Triton X-100 (in PBS) at 37°C for 30 min. The Ana2014 cells were washed once with PBS, and the coverslip was blocked using a blocking buffer (1% BSA in PBS) at 37°C for 1 h. The cells were incubated with the antibodies for RUVBL-1, BoPHB-1, TOMM, TaSP, and affinity-purified antibody TaPHB-1 at 4°C overnight. The cells were washed thrice with PBS and incubated with Alexa Fluor 647-conjugated anti-rabbit IgG (Cell Signaling Technology), Alexa Fluor 555-conjugated anti-mouse IgG (Cell Signaling Technology), and FITC-conjugated anti-IgY (IgY Immunologix) at room temperature for 1 h. The cells were washed thrice with PBS, and the coverslip was mounted on a glass slide with Vectashield antifade mounting medium with DAPI (Vector laboratories). The images were captured using a confocal microscope (Leica SP8, Leica Microsystems) and processed using LAS X software. The images were analyzed with ICY software (47 (link)).