Fitc goat anti mouse igg

The heart sections on the glass slides were de-paraffinized and rehydrated. Antigens were retrieved by microwave, heating the heart sections in sodium citrate–EDTA antigen retrieval solution. The cells were fixed on glass slides and treated with 4% paraformaldehyde. After rinsing with PBS (5 min, thrice), the heart sections and cells were mixed with immunostaining blocking solution for 1 h at 25 °C to prevent nonspecific antibody binding. The heart sections and cells were then incubated with the primary antibody at 4 °C overnight. After washing with PBS (5 min, thrice), the heart sections and cells were incubated with the secondary antibody at room temperature for 1 h. Finally, after washing with PBS (5 min, thrice), the heart sections and cells were stained with DAPI before being imaged using a laser confocal scanning microscope. The following antibodies were used: F4/80 (Santa Cruz Biotechnology), P65 (Cell Signaling Technology), CCKBR (GeneTex, TX), FITC goat anti-mouse IgG (Proteintech, Wuhan, China), Cy3-conjugated goat anti-mouse IgG (Beyotime, Shanghai, China). Alexa FluorTM Plus 647 donkey anti-goat IgG (Invitrogen, Carlsbad), and Cy3-conjugated donkey anti-goat IgG (Beyotime). Images were captured using a fluorescence microscope (Eclipse Ti-U). The images were collected by an investigator who was blinded to the group information of the samples.

B7-H3 expression levels on the surface of human tumor cells were detected using purified mAb-J42 or J42-scFv-Fc. Goat anti-mouse IgG–FITC (Proteintech) was used to label the Fc of mAb-J42. The antibodies used to identify the phenotype of CAR-T cells included CD3-allophycocyanin (APC)-CY7, perforin-APC, CD4-PE, CD8-FITC, TIM3-BV711, and PD-1-BV605 (all purchased from BioLegend). Flow cytometry analysis was performed on a BD Fortessa flow cytometer and analyzed using FlowJo 10.6.0 software.