Immunofluorescence Analysis of ID8-NGL Tumor Tissue

Processing, embedding and sectioning of formalin-fixed ID8-NGL tumor tissue, and hematoxylin and eosin staining for histology, were performed in The Allergy/Pulmonary & Critical Care Med Division Immunohistochemistry Core at Vanderbilt [27 (link)]. Immunofluorescence analysis of formalin-fixed paraffin-embedded tumor tissue or methanol-fixed cultured ID8-NGL cells was performed using standard techniques [21 (link), 19 (link)]. The following primary antibodies: mouse monoclonal anti-pH2AX(ser139) (EMD Millipore, Cat# 05-636, 1:250 dilution); rabbit polyclonal anti-Ki67/Mib-1 (Abcam, Cat# ab16667; 1:200 dilution); and rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Cat# 9661; 1:100 dilution), were used. Secondary antibody used was goat anti-rabbit Alexa Fluor 488 (Life Technologies, Cat# 11070) (all 1:200 dilution). Images were acquired and analyzed as previously described [21 (link), 19 (link)]. For quantifying the percentage of, where applicable, tumor cells or macrophages positive for these proteins, at least 5 independent fields were assessed with at least 200 cells counted per sample.