Alexa fluor 594 conjugated goat anti mouse igg antibody

The serine at position 112 of Yki was changed to alanine by overlapping PCR to generate the Yki-S112A mutant. The open reading frames (ORFs) of P. vannamei Hippo, Yki, and Yki-S112A were cloned into the pAc5.1-GFP vector (54 (link)) to express GFP-tagged proteins. The Wts ORF with a hemagglutinin (HA) tag-encoding sequence was cloned to the pAc5.1/V5-HisA vector (Invitrogen, USA). Drosophila S2 cells at ~80% confluence were transfected or cotransfected with these vectors using FuGENE HD transfection reagent (Promega, USA). At 48 h posttransfection, the expression of Wts-HA was analyzed by immunofluorescence using mouse anti-HA antibody (Cell Signaling Technology [CST], USA) and Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (Abcam, USA). After staining with Hoechst 33342, cells were observed using a confocal laser scanning microscopy at 350/488 nm excitation wavelengths.

RAW 264.7 cells or THP-M cells, respectively, were incubated with FITC-labeled bacteria at a MOI of 100 on Lab-Tek II chamber slides. The trafficking assay was performed as described previously (38 (link)). Briefly, at 30 min incubation, the cells were washed, fixed, permeabilized, and probed with the primary antibody against early endosomal marker EEA1. At 60 min incubation, cells were probed with the antibody against the late endosomal marker, Rab7, or the pre-lysosomal marker, Lamp-1. At 90 min incubation, cells were probed with the antibody against the lysozyme marker, cathepsin D. These mouse monoclonal antibodies (Santa Cruz Biotechnology) were used in dilution 1:300 as recommended by the manufacturer. Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (1:250; Abcam) was used to stain the marker proteins for 1 h. After washing with PBS, the slides were mounted in Vecta shield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector), and viewed with a Fluorescent Microscope Imager A2 with a 100× (1.3 oil) objective (Carl Zeiss AG). The images were obtained and analyzed by Zen 2011 Lite software (Zeiss). Each set was run in triplicate.

Smears of purified synchronized schizont stages of P. falciparum strain 3D7 were fixed with 100% methanol at − 20 °C for 30 min. Samples were permeabilized by treatment with 5% Triton-X-100 overnight and then rinsed with PBS three times at room temperature. Slides were blocked with 3% BSA in PBS overnight at room temperature, followed by incubation with monoclonal antibodies AbMSP10-1, AbMSP10-2, or AbMSP10-3 at 1 µg/mL, for 4 h at room temperature in darkness and then rinsed with PBS three times. AbAMA1 at 1:250 served as a control for schizont development. Then, the slides were incubated with an Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:1000 dilution) (Abcam) and a 1:500 diluted Alexa Fluor 594- conjugated goat anti-mouse IgG antibody (Abcam, Cambridge, MA, USA) for 1 h at room temperature in the dark and rinsed with PBS three times. The slides were mounted with VectaShield DAPI at 1 µg/mL (Vector Laboratories, Burlingame, CA, USA) mounting solution and were covered with cover slides. Parasites treated with monoclonal antibodies were visualized at 365 nm (DAPI, blue), 430 nm (AlexaFluor 488) and 504 nm (AlexaFluor 504) under a confocal microscope, ZEISS LSM 880 (Carl Zeiss, Oberkochen, Germany) at 63× magnification, zoom level 4. Non-infected red blood cells served as a negative control.