Fig. 4

Strongly and weakly stained cases for CDX2 are better separated by fluorescence-based IHC, which has a higher data resolution compared with chromogenic detection using 3,3′-diaminobenzidine (DAB). Three cases illustrating the large variation in CDX2 protein expression among cases with Allred score 8 (a–c). Chromogenic staining with DAB is prone to signal saturation, whereas fluorophores have a much larger linear dynamic signal range enabling DIA to more accurately detect and quantify differential protein expression on a continuous scale. Example of a discrepant case with CDX2 Allred score 8 and DIA score in the lowest quartile showing how cases with clearly reduced protein expression can be scored as strong because the dynamic signal range of DAB is not sufficient to differentiate both the weak and the strong cases, leading to some of the weaker cases being stained too strong to be readily separated by visual analysis (d). Single-cell analysis (right image column) furthermore enables a more accurate and objective scoring of cases with large variation in protein expression between individual cells, here illustrated by the gradual difference in CDX2 expression from left to right on the histospot which is less noticeable for the DAB stain. CDX2 signal intensities were binned into 10th percentiles on a cell-by-cell basis. Bin1 corresponds to the lower percentile (blue colour) and Bin10 (dark red colour) to the higher percentile. Fluorescent images are scaled relative to each other; hence CDX2 staining in A appears oversaturated due to the relatively much higher protein expression in this sample. Scale bar, 0.1 mm