Anti mouse igg1 fitc

Attached cells were fixed by 4% paraformaldehyde for 30 min, and penetrated by Triton X-100 for 15 min, then blocked by BSA for 30 min. DYKDDDK Tag (9A3) mouse mAb (1: 100, Cell signaling) was incubated at room temperature for 1 h. After rinsed by PBS, Anti-mouse IgG1 FITC (1:100, eBioscience) was incubated for 30 min. Slides were mounted by VECTASHIELD Mounting Medium with DAPI (Vector Labs). Images were captured by confocal microscope, with Zeiss LSM Image Examiner (Carl Zeiss).

Tissue samples were fixed in 4% formaldehyde, processed, embedded in paraffin and sectioned with Leica Microtome. Sections were stained using Ki67 antibody (Abcam) and CD31 antibody (Abcam). For each sample three randomly chosen fields were imaged by Leica camera and analyzed by ImageJ software. Cryo-sections (5 μm thick) were incubated with LAMP-1 primary antibody (Santa Cruz Biotechnology) followed by the Anti-Mouse IgG1 FITC (eBioscience) secondary antibody. Verteporfin auto-fluorescence was observed in the far-red spectrum. Coverslips were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, H-1500) and fluorescence images were taken using an automated inverted microscope (Leica DMSI4000 B).

The anti-human B7-H3-PE mAb and mouse IgG1-PE isotpye antibody were purchased from R&D System. The anti-human CD3-FITC, anti-mouse IgG1-FITC, and anti-mouse IgG2a-FITC secondary antibodies were provieded by eBioscience. The cells were assayed with a Guava flow cytometrometer Easycyte (Guava, Hayward, CA, USA) and the data analysis was carried out with the FlowJo Software Version 7.6.1.