In the pursuit of reducing re-excision rates in breast conserving surgery, a dual probe specimen staining technique has emerged as a promising approach to identify positive margins during surgery. This approach generally involves staining the tissue with a fluorescent dye targeted to a biomarker of interest, such as a cell surface receptor, and an untargeted counterpart, imaging both dyes and using the two images together to compensate for instrumentation inhomogeneities and non-specific uptake. A growing body of literature suggests that this approach can effectively discriminate tumor and normal tissue in gross fresh specimens in reasonable timeframes. However, the robustness of the staining protocol is still under investigation as all parameters have not been fully evaluated. In this paper, we examine the effect of staining temperature on diagnostic performance. Tumor (overexpressing EGFR) and normal fresh specimens were stained at room temperature or 37 ° C and diagnostic performance compared using area under the curve (AUC) from receiver operator characteristic (ROC) analysis. The results suggest that the use of Licor IRDye800CW-labeled anti-EGFR antibody and Licor IRdye680RD-labeled control antibody as the probe pair is not significantly affected by staining temperature, in contrast to our experience with quantum-dot labeled antibodies. The robustness of the technique using these stains is reassuring and simplifies the staining protocol.