Alexa fluor 647 labeled goat polyclonal anti rabbit igg antibody

Tumor specimens in paraffin‐embedded blocks were cut into 4‐µm‐thick sections. Nonspecific binding was blocked using nonspecific staining blocking reagent (Dako). The sections were incubated with rabbit monoclonal anti‐CD103 antibody (clone: EPR4166 (2); 1/1000; Abcam), mouse monoclonal anti‐CD8 antibody (clone: C8/144; 1/250; Dako), mouse anti‐CD20 antibody (clone: L26, prediluted; Dako, Agilent Technologies, Inc) and mouse anti‐CD11c antibody (cat. no. ab215858; 1/100; Abcam) at 4°C overnight. The sections were subsequently incubated with Alexa Fluor 488‐labeled goat polyclonal anti‐mouse IgG antibody (cat. no. ab150113; 1/1000; Abcam) and Alexa Fluor 647‐labeled goat polyclonal anti‐rabbit IgG antibody (cat. no. ab150079; 1/1000; Abcam) for 1 h at room temperature. The sections were covered with glass using ProLong™ Gold antifade reagent with DAPI (cat. no. P36935; Invitrogen/Thermo Fisher Scientific). Digital images were taken with an all‐in‐one fluorescence microscope (BZ‐8000; Keyence).

Human specimens of metastatic LNs in paraffin‐embedded blocks were cut into 4‐μm‐thick sections. After heat‐mediated antigen retrieval and immunohistochemical analysis, nonspecific binding was blocked using nonspecific staining blocking reagent (Dako). The sections were incubated with rabbit monoclonal anti‐CD103 antibody (clone: EPR4166(2); 1/1000: Abcam), mouse monoclonal anti‐CD8 antibody (clone: C8/144B; 1/250: Dako), mouse monoclonal anti‐granzyme B antibody (cat. no. sc‐8022; 1/100; Santa Cruz Biotechnology), and mouse monoclonal anti‐CD11c antibody (cat. no. ab215858; 1/100; Abcam) at 4°C overnight. The sections were subsequently incubated with Alexa Fluor 488‐labeled goat polyclonal anti‐rabbit IgG antibody (cat. no. ab150113; 1/1000: Abcam) and Alexa Fluor 647‐labeled goat polyclonal anti‐rabbit IgG antibody (cat. no. ab150079; 1/1000: Abcam) for 1 h at room temperature. Prolong Gold antifade reagent with DAPI (cat. no. P36935; Invitrogen/Thermo Fisher Scientific) was then added to the sections and covered with glass coverslips. Digital images were taken using an all‐in‐one fluorescence microscope (BZ‐8000: Keyence).