Alexa fluor 555 conjugated goat anti rabbit igg h l

HzAM1 cells were infected with vHaBac-egfp-ph, vHaBacΔpifs-Acpifs-ph or vHaBacΔpifs-Sppifs-ph at an MOI of 5. At 72 h p.i., cells were fixed with 4% paraformaldehyde and made permeable with 0.2% Triton X-100. After being blocked with 5% BSA, the cells were incubated with anti-PIFs polyclonal antibodies and then with Alexa Fluor^™ 555-conjugated Goat Anti-Rabbit IgG H&L (Abcam) as the secondary antibody. Nuclei were stained with Hoechst stain. The subcellular localization of the PIFs was detected by fluorescence microscopy.

Immunofluorescence was conducted for staining HSP, actin, and the nucleus. HSP was stained with Anti-HSP 70 (ADI-SPA-810-F; Enzo Life Sciences, Inc., Farmingdale, NY, USA) and Alexa Fluor 555-conjugated goat anti-rabbit IgG H&L (ab150078; Abcam, Cambridge, UK). Actin and the nucleus were stained with ActinGreen 488 Ready Probes reagent (R37110; Thermo Fisher Scientific Inc.) and Hoechst 33342 (H1399; Thermo Fisher Scientific Inc.). Staining was performed by the following procedure. First, 4% paraformaldehyde (09154-56; Nacalai Tesque, Inc., Kyoto, Japan) supplemented with Triton X-100 (C0875; Sigma-Aldrich) was applied to the cells, which were then incubated for 8 min at room temperature for fixation and permeabilization. Then, after blocking nonspecific binding sites with ImmunoBlock (CTKN001; KAC Co., Ltd., Kyoto, Japan) for 1 h at room temperature, ActinGreen 488 Ready Probes reagent and Hoechst 33342 were applied. Subsequently, the cells were incubated with 2 µL/1 mL Anti-HSP 70 for 1 h at room temperature. Then, they were incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG H&L for 30 min.