Alexa fluor 488 conjugated f4 80

Formalin-fixed, paraffin-embedded sections were prepared using femoral artery 7 days after cuff placement. Endogenous peroxidase was blocked by incubation in 3% H₂O₂ for 15 min, and nonspecific protein binding was blocked by incubation for 10 min in Blocking Reagent (Nichirei Bioscience Inc., Tokyo, Japan). The sections were incubated overnight at 4°C with the primary antibody, anti-proliferating cell nuclear antigen (PCNA) antibody (Novocastra Laboratories, Ltd., Newcastle upon Tyne, UK), and phosphorylated extracellular signal-regulated kinase (ERK) and total ERK antibodies (Cell Signaling Technology, Pickering, ON) and Alexa Fluor 488-conjugated F4/80 (BioLegend Inc., San Diego, CA) and R-PE-conjugated LY-6G/-6C (Hycult Biotech Inc., Plymouth Meeting, PA). Antibody binding was visualized by followings: 1) a Zeiss Axioskop2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with a computer-based imaging system for 3, 3’-diaminobenzidine (DAB) staining using a detection kit, Histofine (Nichirei Bioscience Inc.) for PCNA and ERK staining, and 2) a fluorescence microscope (Keyence BZ-9000, Osaka, Japan) equipped with a computer-based imaging system for immunofluorescent staining.

Iliac lymph nodes were excised from immunized mice and immediately immersed in Hank's Balanced Salt Solution medium and put on ice. Lymph nodes were then treated with 1 mg/mL Collagenase D (Roche Diagnostics, Indianapolis, IN) at 37 °C for 20 min to release cells. Cells were washed with PBS containing 2 mM EDTA, blocked with 1% normal rabbit serum, and stained with the following antibodies and reagents: Zombie Violet, biotinylated anti-Ly6G, biotinylated anti-Ly6C, Alexa Fluor 488 or PerCP-Cy5.5-conjugated anti-CD11c, PE-conjugated anti-I-A/I-E, PE/Cy7-conjugated anti-CD86, Alexa Fluor 488-conjugated F4/80 (all from BioLegend, San Diego, CA), and PE-conjugated anti-Siglec F (BD Bioscience, San Jose, CA). Biotinylated antibodies were detected with Alexa Fluor 488 or APC-conjugated streptavidin. After staining, cells were fixed in 2% paraformaldehyde. Cells were examined in a Canto II flow cytometer (BD Bioscience, San Jose, CA), and data were analyzed with FlowJo software (FlowJo, Eugene, OR).