Quantifying Hypoxia-Induced pSMAD Signaling

Immunofluorescence staining for pimonidazole was performed as previously described [39 (link)] on 5µm thick xenograft cryosections. Tumor sections were double stained for pimonidazole and pSMAD2 or pSMAD3 antibody (1:50). For quantitation of pSMAD staining intensity, a minimum of 6 representative areas from at least 3 different tumors for each condition were captured using an Axioskop 2 phase-contrast/epifluorescence microscope (Carl Zeiss Inc., Oberkochen, Germany). Fluorescence intensities were analyzed using the ImagePro Plus software version 6 (Media Cybernetics, Rockville, MD, USA). The sum of pSMAD labeling intensity (density) relative to the total area of pimonidazole positive or negative areas was calculated.