Alexa fluor 594 conjugated secondary ab against rabbit igg

The anti-FLAG mouse monoclonal antibody (number F3165), anti-pTurboGFP rabbit polyclonal antibody (number AB513), and anti-GAPDH mouse monoclonal antibody (number 60004-1-Ig) were purchased from Sigma-Aldrich, Everogen, and Proteintech, respectively. The anti-NS38 polyclonal mouse antibody and anti-NS80 polyclonal rabbit antibody against the GCRV-873 strain were prepared previously (7 (link)– (link)10 (link)). Lovastatin (S2061), VitD (S1466), MG132 (S2619), and 3-MA (S2767) were purchased from Selleck (Shanghai, China). Goat-anti-mouse immunoglobulin-horseradish peroxidase (Ig-HRP) conjugate secondary antibody, goat-anti-rabbit Ig-HRP conjugate secondary antibody, Alexa Fluor 488-conjugated secondary Ab against mouse IgG, Alexa Fluor 594-conjugated secondary Ab against rabbit IgG, 4′,6-diamidino-2-phenylindole (DAPI), Subcellular Protein Fractionation kit, Lipofectamine 2000, and protease inhibitor cocktail were purchased from Thermo Fisher Scientific. The FLAG immunoprecipitation kit and chloroquine (C6628) were purchased from Sigma-Aldrich.

After adhering RAW264.7 cells (4 × 10⁵ cell/dish) to 35-mm glass-bottom dishes for 1 h, cells were incubated with Alexa Fluor 488-labelled AGEs (200 ng/ml) for 4 h. Then, cells were washed with PBS (−) and fixed with 4% paraformaldehyde (PFA) at room temperature for 30 min followed by washing with PBS-T (PBS pH 7.6 with 0.1% Tween-20). Subsequently, cells were incubated in blocking buffer containing 1% BSA, 0.3 M glycine in PBS-T at room temperature for 1 h to permeabilise the cell membrane and block non-specific protein-protein interactions. Cells were next incubated with normal rabbit IgG (1:100 dilution for negative control) or Ab directed against CD204 (1:100 dilution, PA5-22957, Thermo Fisher Scientific) in blocking buffer at 4 °C overnight. After rinses in PBS-T, cells were incubated with Alexa Fluor 594-conjugated secondary Ab against rabbit IgG (1:500 dilution, A-11037, Thermo Fisher Scientific) at room temperature for 1 h. Then, cells were washed with PBS-T and photomicrographs were taken at 0.5–0.8-μm intervals for the z-axis at original magnification × 400 with a confocal laser C2 microscope.