Quantifying Regulatory T Cells in Tumor Samples

Tregs were stained by using a FoxP3 mouse monoclonal antibody clone 236A/E7 (eBioscience, San Diego, CA, USA) at a dilution of 1:200. After deparaffinization with xylene and rehydratation with decreasing concentrations of ethanol, epitope retrieval was performed by immersing the samples in citrate buffer (ScyTek, Logan, UT, USA), and then by heating in micro-waves. Primary antibody was incubated during one hour at room temperature followed by a HRP-mouse secondary antibody, as recommended in the manufacturer’s protocol (CSAII kit, Dako, Glostrup, Denmark). Next, sections were reacted with phenol/fluorescyl-tyramid amplification reagent and HRP-tertiary antibody. Finally, diaminobenzidine and hydrogen peroxide were added on each tumor slide. The number of Foxp3+ cells (nuclear staining) was counted in 5 fields in each area (stromal and intra-tumoral compartments) with an Axio-Cam MRC5 optical microscope (Zeiss, Hallbergmoos, Germany) at 400× magnification. The mean of these 5 fields was then calculated and the median number of FoxP3+ cells was established for each compartment. Finally, we used the Cutoff Finder web application [22 (link)] to calculate the optimal cutoff points for stromal and intra-tumoral FoxP3+ Treg populations.