Cy3 conjugated affinipure goat anti mouse igg h l

Plasmids containing overexpressed sequences were digested by restriction endonucleases (Thermo Fisher, USA) and connected to the insect expression plasmid PIZT-V5/His with T4 ligase (TAKARA, China). The plasmid was extracted with an E.Z.N.A.® Endo-Free Plasmid Mini Kit II (Omega, USA), and FuGENE® HD Transfection Reagent (Promega, USA) was used to transfect the cells. After 24 h, Sf9 cells were treated with 0.05 mM harmine (98%, Bidepharm, China), and cell immunofluorescence was carried out. The primary antibody was Sf-Atg8 polyclonal antibody (1:1000, MF069154.1, GeneCreate, Wuhan, China) and Sf-Cathepsin L polyclonal antibody (1:1000, HQ110065.1, laboratory-owned), and the secondary antibody was CY3 Conjugated AffiniPure Goat Anti-mouse IgG (H+L) and CY3 Conjugated AffiniPure Goat Anti-rabbit IgG (H+L) (Boster, China). The images were taken by a laser confocal microscope (Nikon A1, Japan) and the fluorescence analysis were carried out with the built-in Nikon software.

The RNAi procedure was performed with reference to the longer double-stranded RNA (dsRNA) synthesis step in the T7 RiboMAX™ Express RNAi System (Promega, USA). The obtained dsRNA was transfected into Sf9 cells after overnight culture and then treated with 0.05 mM harmine for 24 h. cell immunofluorescence was carried out and the primary antibody, Sf-Atg8 polyclonal antibody and Sf-Cathepsin L polyclonal antibody, were used. The secondary antibody was CY3 Conjugated AffiniPure Goat Anti-mouse IgG (H+L) and FITC Conjugated AffiniPure Mouse Anti-Rabbit IgG (H+L) (Boster, China). The images were taken by an inverted fluorescence microscope (Nikon 600 L, Japan) and merged with the built-in Nikon software. Fluorescence statistics were performed using Image-Pro Plus 6.0 software (Media Cybernetics, USA) and area, diameter (mean), density (mean), and IOD were evaluated. The Cathepsin L-positive points of red fluorescence were selected with intensity ≥15, and the Atg8-positive points of green fluorescence were selected with intensity ≥20.