Fitc conjugated goat anti mouse antibody

The sequencing results of gp85 gene revealed that SD20LH01 was belonged to ALV-K. To further verify the isolated virus, immunofluorescence assay (IFA) experiments were performed. The cell supernatant was discarded, and infected DF-1 cells were fixed with paraformaldehyde at 4°C overnight. After that, permeabilized with 0.25%Triton X-100 for 15 min at room temperature. Then, cells were washed 3 times with PBS and incubated with mouse anti ALV-K gp85 polyclonal antibody for 1 h at 37°C. Next, a FITC-conjugated goat anti-mouse antibody (Abcam, UK) was used as the secondary antibody for 1 h at 37°C. Finally, the cells were washed 3 times again with PBS and observed under a fluorescence microscope. The mouse anti ALV-K gp85 polyclonal antibody was prepared in our laboratory. Briefly, ALV-K gp85 gene of JS11C1 strain was amplified and inserted into pET32-a (+) prokaryotic expression vector, and transformed into BL21 E. coli cells to obtain the recombinant protein. Mouse were immunized with the purified recombinant protein mixed with Freund's complete adjuvant to prepare the anti-ALV-K gp85 serum.

The slides were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 (Beyotime Biotechnology) for 30 min. After blocking in 1% BSA for 15 min at room temperature, cells were incubated with primary antibody overnight at 4 °C. The primary antibodies were as follows: γH2AX antibody (1:200 diluted, Thermo Fisher Scientific), USP43 antibody (1:50 diluted, Santa Cruz Biotechnology), HDAC2 antibody (1:100 diluted, Proteintech Group) and β-catenin antibody (1:50 diluted, Proteintech Group). Then cells were incubated with Cy3-conjugated goat anti-rabbit antibody (1:200 diluted, Invitrogen), Cy3-conjugated goat anti-mouse antibody (1:200 diluted, Invitrogen) or FITC-conjugated goat anti-mouse antibody (1:200 diluted, Abcam) for 60 min at room temperature in the dark. After washing with PBS, cells were counterstained with DAPI (Aladdin) to mark the nuclei. The slides were sealed with fluorescent mounting medium (Solarbio Science), and observed under the microscope.

Liver issue slices embedded in paraffin (5-μm thick) were prepared for immunofluorescence analysis. The TLR4 antibody (1:50, Thermo Invitrogen, Shanghai, China), CD31 antibody (1:50, Abcam, Shanghai, China), PTRF antibody (1:50, Thermo Invitrogen, Shanghai, China), and α-SMA antibody (1:50, Abcam, Shanghai, China) were used as primary antibodies, and the FITC-conjugated goat anti-mouse antibody (1:500, Abcam, Shanghai, China) and CY3-conjugated goat anti-rabbit antibody (1:500, Abcam, Shanghai, China) were used as the secondary antibodies; 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear indicator. The slides were subsequently visualized using a confocal laser-scanning microscope (LSM 880, Carl Zeiss, Oberkochen, Germany).

MCs were collected and fixed utilizing the 4% paraformaldehyde on microslide for 10 min, which were further treated with triton-100 for 15 min. Following incubating with 5% BSA, cells were added with the mouse anti rat CK18 antibody (1:1000, biorbyt, Wuhan, China) at 37°C for 1.5 h, which were further added with the solution diluted with FITC Conjugated goat anti mouse antibody (1:500, Abcam, Cambridge, USA) at 37°C for 45 min. Lastly, the slides were sealed using nail polish after adding the DAPI solution. The fluorescence was observed under the fluorescence microscope (KEYENCE, Tokyo, Japan).

DF-1 cells were harvested at 48 hpi, washed thrice gently with D-PBS, fixed with 4% paraformaldehyde for 15 min, blocked with QuickBlock Blocking Buffer for Immunol Staining (Beyotime Biotechnology, Shanghai, China), incubated with mouse anti-ENV antibody (mAb JE9), washed thrice with Phosphate-Buffered Saline with Tween 20 (PBST), incubated with FITC-conjugated goat anti-mouse antibody (Abcam, United Kingdom), washed thrice with PBST again, and then incubated with DAPI for 5 min before visualizing the expression levels of viral protein in DF-1 cells using fluorescence microscopy (Nikon, 200×).