Pimonidazole immunohistochemistry

Sections were cleaned and rehydrated according to standard procedures. Monoclonal antibody directed against pimonidazole (included in the Hypoxyprobe-1 green kit) was used for immunohistochemical staining of the tissue sections. Fluorescence microscopy was performed using an Olympus BX51WI equipped with a high resolution digital CCD ORCA-285 (Hamamatsu Corp., Hamamatsu city, Japan) illuminated with a mercury burner and the appropriate fluorescent cubes (XF100-2 and XF02-2, Omega Optical, Brattleboro, VT). Images for pimonidazole antibody-stained areas and Hoechst were prepared using Wasabi Imaging Software (Hamamatsu Corp). The ratio of pixels stained for pimonidazole in each region, to the total cellular area of the image was calculated. Ten images were analyzed by section, and the results were pooled to determine the mean and standard deviation. To indicate the co-localization of pimonidazole and Hoechst in cell, images were superimposed.