Anti iba1

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)}

Animals were sacrificed 1-day post-MCAO, and their brains were fixed in 4% paraformaldehyde by transcardiac perfusion and then post-fixed in the same solution overnight at 4 °C. Brain sections (20 μm) were produced using a vibratome, and immunological staining was performed as previously described [42 (link)]. Primary antibodies were incubated after diluting to 1:500 for anti-ionized calcium-binding adaptor molecule-1 (Iba1; Wako Pure Chemicals, Osaka, Japan) and to 1:250 for anti-Mac-2 (Abcam, Cambridge, UK). After washing with PBS containing 0.1% Triton X-100, the sections were incubated with anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) for anti-Mac-2 or anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) for anti-Iba1 in PBS at room temperature for 1 h and visualized using the HRP/3,3′-diaminobenzidine (DAB) system (Vector Laboratories, Burlingame, CA, USA).