Immunohistochemical Analysis of Tumor Microenvironment

The tumors were collected, frozen in liquid nitrogen, and sectioned into 5 μm slices. Macrophages in the tumor sections were stained with anti-F4/80 and anti-CD206 antibodies (Abcam, Cambridge, UK), followed by appropriate fluorochrome-conjugated (Alexa Fluor 594 (Abcam) and FITC (Vector Laboratories, Burlingame, CA, USA) secondary antibodies. The cytotoxic T lymphocytes were stained with anti-CD8α (Abcam) and subsequently with Alexa Fluor 594-conjugated secondary antibodies (Abcam). For the immunohistochemical analyses of pericytes coverage, tumor blood vessels sections were incubated with anti-α-Smooth Muscle Actin and anti-CD31 antibodies (Abcam) and subsequently with Texas Red and FITC-conjugated secondary antibodies (Vector Laboratories) [13 (link)]. All the sections were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Microscopic observations were performed using a LSM710 confocal microscope (Carl Zeiss Microscopy GmbH). The obtained confocal images were analyzed with ImageJ 1.48v (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, LOCI, University of Wisconsin, USA) and the results were expressed as the percentage of area [%].