Immunostaining for Ki-67 (Vector), HER2 (AB16901; Abcam), and STAT1 (AB47425; Abcam) was performed as previously described6 (link). Secondary antibodies (AF488 AffiniPure donkey anti-mouse IgG, Cy3 AffiniPure donkey anti-rabbit IgG) were from Jackson ImmunoResearch Laboratories. Images were acquired with a Yokogawa spinning disk confocal on an inverted Nikon Ti fluorescence microscope using MetaMorph image acquisition software, and 3–5 fields were analyzed per tumor. Image analysis was performed using a semi-automated in-house platform (NIH ImageJ).
Immunofluorescence for FoxP3 (clone FJK-16s; eBioscience), CD8 (clone 4SM15; eBioscience), and Ki-67 (clone SP6; Thermo Scientific) was performed as previously described21 (link). Secondary antibodies (AF488 donkey anti-rabbit IgG, AF647 goat anti-rat IgG) were from Life Technologies. Tissues were counterstained with DAPI (Invitrogen). Images were acquired on a Nikon Eclipse Ni microscope using NIS Elements software, and 5–10 fields were analyzed per tumor.
Immunofluorescence for FoxP3 (clone FJK-16s; eBioscience), CD8 (clone 4SM15; eBioscience), and Ki-67 (clone SP6; Thermo Scientific) was performed as previously described21 (link). Secondary antibodies (AF488 donkey anti-rabbit IgG, AF647 goat anti-rat IgG) were from Life Technologies. Tissues were counterstained with DAPI (Invitrogen). Images were acquired on a Nikon Eclipse Ni microscope using NIS Elements software, and 5–10 fields were analyzed per tumor.
