Immunofluorescence Analysis of Tumor Microenvironment

Immunofluorescence (IF) staining of tumor tissue harvested from the Group I [²²⁵Ac]αMSH-PEG-Cy5-C′ dot-treated mice and Group III vehicle-treated mice (see Pharmacodynamic Studies section above) was performed to image the kinetics of immune cell in the TME post-treatment. Representative animals were euthanized at 1, 24, 96, and 120 h post-treatment and harvested tumor was fixed in 4% paraformaldehyde/PBS for 24 h. Fixed tissue was paraffin embedded and cut into 5-μm sections and mounted for imaging. IF staining was performed at the MSKCC Molecular Cytology Core Facility using a Discovery XT processor (Ventana Medical Systems). Stains used include anti-CD3 (0.5 μg/mL, #A0452; eBioscience) and anti-IBA1 (0.4 μg/mL, #091-19741; Vector). Tumor tissue sections were scanned using a Mirax digital slide scanner (Carl Zeiss Microimaging) with the × 20 lens and analyzed with Pannoramic Viewer software and quantification of areas stained positive for T cells and macrophages was performed using Fiji imaging software.^{33 ,34 (link)}