Supplemental Data:

Table 1S. Aperio nuclear quantification algorithms for single and double immunostaining

Parameters / Single Stain / Double Stains / Parameters-continue / Single Stain / Double Stains
Version / 9.1 / 9.1 / Min Compactness / 0.1 / 0
View Width / 1000 / 1000 / Min Elongation / 1.00E-02 / 0.1
View Height / 1000 / 1000 / Remove Light Objects / 0 / 0
Overlap Size / 100 / 100 / Weak(1+) Threshold / 210 / 210
Image Zoom / 1 / 1 / Moderate(2+) Threshold / 188 / 188
Classifier / None / None / Strong(3+) Threshold / 140 / 140
Classifier Neighborhood / 0 / 0 / Black Threshold / 0 / 0
Pixel Size (um) / 0.4983 / 0.4983 / Edge Trim / None / Weighted
Averaging Radius (um) / 1 / 0.5 / Markup Image Type / Analysis / Analysis
Averaging Radius (Pixels) / 2 / 1 / Nuclear Red OD / 0.691 / 0.696858
Curvature Threshold / 2.5 / 6 / Nuclear Green OD / 0.639 / 0.643073
Segmentation Type / 1 / 2 / Nuclear Blue OD / 0.335 / 0.317563
Threshold Type / 1 / 1 / Positive Red OD / 0.396 / 0.327
Lower Intensity Threshold / 0 / 0 / Positive Green OD / 0.588 / 0.641
Upper Intensity Threshold / 200 / 230 / Positive Blue OD / 0.704 / 0.695
Min Nuclear Size (um^2) / 2 / 20 / Color(3) Red OD / 0 / 0.133
Min Nuclear Size (Pixels) / 8 / 81 / Color(3) Green OD / 0 / 0.742
Max Nuclear Size (um^2) / 1.00E+06 / 1.00E+06 / Color(3) Blue OD / 0 / 0.657
Max Nuclear Size (Pixels) / 4.03E+06 / 4.03E+06 / Clear Area Intensity / 240 / 240
Min Roundness / 1.00E-02 / 0.1

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