Fitc conjugated donkey anti mouse igg

Immunofluorescence analysis for asexual stages, gametocytes, ookinetes, and sporozoites was carried out by fixing the cells with 4% paraformaldehyde and 0.0075% glutaraldehyde, followed by permeabilization with 0.1% Triton X-100 and subsequent treatment with 0.1 M glycine. Blocking was performed with PBS containing 2% BSA for 3 h. The incubation for primary antibodies was carried out in the same blocking buffer for 6 h, followed by the addition of secondary antibodies for 3 h^{77 (link)}. For the oocyst, the mosquito gut was fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X-100, followed by blocking in 4% BSA^{78 (link)}. For exo-erythrocytic stages, HC-04 cells infected with sporozoites were fixed with 4% paraformaldehyde, permeabilized with 0.01% Triton X-100 and blocked with PBS containing 1% BSA^{79 (link)}. Parasite GS-specific polyclonal sera were used at 1:250 dilution. Anti-UIS4 antibody (Origene, AB0042-200) was used at 1:1000 dilution. FITC-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific, A24501) was used at 1:250 dilution. Rabbit anti-goat AF594 (Thermo Fisher Scientific, A-11080) was used at 1:400 dilution. Images were captured with 20x/60x/100x objectives using Olympus IX83 microscope with DP73 high-performance camera.

Immunostaining analysis was conducted as previously described [27 (link), 32 (link), 33 ]. Briefly, TGs were sectioned (15-μm thickness) on a cryostat (Leica CM1950), permeabilized with Triton X-100, and blocked with 5% goat serum. After washing, TG sections were incubated with primary antibodies against adipoR1 (rabbit, 1:300, Abcam), NeuN (mouse, 1:500, Cell Signaling Technology), GS (mouse, 1:500, Abcam), NF200 (mouse, 1:500, Sigma‒Aldrich), and CGRP (mouse, 1:300, Abcam) and visualized using IB₄-fluorescein isothiocyanate (FITC) (Sigma‒Aldrich) or the appropriate secondary antibodies, including FITC-conjugated donkey anti-mouse IgG (1:300, ThermoFisher Scientific) and Cy3-conjugated donkey anti-rabbit IgG (1:300, Merck Millipore). Fluorescence images were captured utilizing a Nikon104c microscope equipped with a CoolSnap-ProColor CCD camera (Photometrics).

Immunofluorescence analysis of translocation was conducted as described in our previous studies [26 (link)]. In brief, TG neurons were treated with 100 nM adiponectin for 30 min and then fixed with 4% PFA. After incubation with PBS containing 10% goat serum and 0.2% Triton X-100, neurons were probed with mouse anti-PKCβ1 (1:500, Abcam) overnight at 4 °C and visualized with FITC-conjugated donkey anti-mouse IgG (1:300, ThermoFisher Scientific). The images were captured with a Zeiss LSM510 confocal microscope and analyzed with Image-Pro Plus v6.0 analysis software (Media Cybernetics).