Cy3 conjugated goat anti rabbit igg h l

Immunofluorescence Assay was used to detect the NcROP5 subcellular localization as previously described (Li et al., 2016 (link)). Appropriate numbers of parasites were coated on glass coverslips in 12-well plates and then fixed in 4% paraformaldehyde, permeabilized with 0.25% Triton X-100 and blocked with 3% BSA. Subsequently, the cells were incubated with a mouse anti-HA monoclonal antibody, mouse anti-NcROP5 and rabbit anti-NcSRS2 antibody (National Animal Protozoa Laboratory in China Agricultural University) followed by FITC-conjugated goat-anti mouse IgG (H+L), Cy3-conjugated goat-anti mouse IgG (H+L), and Cy3-conjugated goat-anti rabbit IgG (H+L) (Sigma, USA). The nuclear DNA was stained with Hoechst33258 (Sigma, USA). Extracellular iΔNcROP5 parasites were identified by IFA. The images were obtained using a Leica confocal microscope system (Leica, TCS SP52, Germany).

Immunofluorescence assays (IFAs) were performed as described previously [13 ]. Briefly, tachyzoite-infected HFFs were fixed with 4% paraformaldehyde. After three washes with PBS, the cell membranes were permeabilized and samples were blocked by incubation with 0.25% Triton X-100/PBS and 3% bovine serum albumin for 30 min at room temperature. The primary antibodies used were rabbit or mouse anti-FLAG (1:50; Sigma-Aldrich, St. Louis, MO, USA), rabbit or mouse anti-HA (1:50; Sigma-Aldrich) and rabbit anti-GAP45 (1:200). 4′,6-Diamidino-2-phenylindole (DAPI; 1:100; Sigma-Aldrich) and secondary antibodies (FITC-conjugated goat anti-mouse IgG [H+L], 1:50; Cy3-conjugated goat anti-rabbit IgG [H+L], 1:100) were incubated together. Images were obtained using a Leica confocal microscope system (TCS SP52; Leica Microsystems, Wetzlar, Germany).
For the immunoblotting assays, 1 × 10⁷ parasites were collected and purified by filtration through a 5-µm filter membrane and lysed with RIPA buffer (Huaxinbio, Beijing, China). The primary antibodies used were rabbit or mouse anti-FLAG (1:500; Sigma-Aldrich), rabbit or mouse anti-HA (1:500; Sigma-Aldrich), mouse anti-actin (1:5000). Horseradish peroxidase-conjugated antibodies were used as secondary antibodies (1:5000; Invitrogen, Thermo Fisher Scientific).

The RHΔku80 strain was used as the parental parasite. The Δdsk2a strain (the RHΔku80 strain lacking the dsk2a gene) were also used. These two strains were preserved in our laboratory.
Parasites were cultured in human foreskin fibroblast cells (HFFs) (ATCC, Rockefeller, MD, USA) growing in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO₂.
Primary antibodies used in the study: rabbit anti-ubiquitin monoclonal Ab (ab134953) was purchased from abcam (Cambridge, UK), mouse anti-HA MAb was purchased from Sigma (St. Louis, MO, USA), and mouse anti-IMC1, anti-ATG8 and anti-actin, and rabbit anti-GAP45, anti-SAG1, anti-ENR and anti-centrin1 polyclonal antibodies were preserved in our laboratory.
Secondary antibodies used in the study: FITC-conjugated goat anti-mouse IgG (H + L) and goat anti-rabbit IgG (H + L), Cy3-conjugated goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) horseradish peroxidase (HRP) were purchased from Sigma (St. Louis, MO, USA).