Alexa fluor 546 conjugated goat anti rabbit igg h l

CAPE (purity ≥98%) was obtained from Nature Standard (Shanghai, China). Concanavalin A type IV (ConA), lipopolysaccharide (LPS), Percoll, Triton X-100, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Recombinant mouse IL-12 (rIL-12; p70); purified Ultra-LEAF™ anti-mouse IL-4 antibody; purifiedLEAF™ anti-mouse CD3 antibody; purified LEAF™anti-mouse CD28 antibody; antibodies against mouse CD4, IL-17A, IFN-γ, Foxp3, CD25, and CD69; cell activation cocktail; brefeldin A solution; fixation/intracellular staining permeabilization wash buffer; and Foxp3 fixation/permeabilization buffer were obtained from BioLegend. RPMI 1640 medium, fetal bovine serum (FBS), and GlutaMAX were obtained from Gibco. Primary antibody against Iba-1 was purchased from Wako Pure Chemical Industries. Antibody against NF-κB p65 was obtained from Santa Cruz, and myelin basic protein (MBP) was purchased from Proteintech. Alexa Fluor 546-conjugated goat anti-rabbit IgG (H + L) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) antibodies were purchased from Invitrogen. A haematoxylin-eosin (HE) staining kit and cell counting kit (CCK)-8 were purchased from Beyotime Biotechnology (Shanghai, China). Human XL Cytokine Array Kit was obtained from R&D.

T. gondii RH strain was propagated and lysed by passing the cells though a #27 syringe and was filtered by using a 5-μm filter. The lysate was subjected to immunoblotting as described elsewhere [30 (link)]. For indirect immunofluorescence microscopy (IFA), purified extracellular tachyzoites were fixed on a 14-well slide with 40 % formaldehyde and were permeabilized with 0.1 % Triton X-100. The slide was incubated with antibodies against TgCaMKrk and GRA6, TgGAP45, ROP-1, or M2AP, respectively, for 60 min at 37 °C and then rinsed three times with PBS. After incubation with AlexaFluor 488-conjugated goat anti-mouse IgG (H + L) and AlexaFluor 546-conjugated goat anti-rabbit IgG (H + L) (Invitrogen) for 60 min at room temperature, the slide was rinsed three times with PBS. Intracellular tachyzoites were prepared by infecting Vero cells cultured in an eight-well chamber slide for 48 h; the slide was then fixed and stained as described above.