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Kimball Et Al 2019, Figure 2
Kimball Et Al 2019, Figure 2
DAPI
X
CD3 CD4 B220
FoxP3 CD8 F4/80
Autofluorescence Signal No signal
ex: 520 single stain
CD3 Examine signal across
FoxP3
CD4
CD8
Assign IDs and 4. Confirm signals make biological sense all channels to identify
B220 pseudocolors spectral bleedover
F4/80
DAPI for each stain
Autofluorescence Unexpected colocalization
of markers suggests
Utilize appropriate single
staining artifacts and/or
stain, unstained, and DAPI
issues with panel
controls for spectral unmixing F4/80 CD3
design/staining protocol
Change settings while Unsegmented Image Fill holes Fill holes Fill holes Fill holes Fill holes
visualizing the results Roundness Roundness
Min-Max size
Roundness Roundness
Min-Max size
Roundness
Min-Max size Min-Max size Min-Max size
of segmentation Refine splitting Refine splitting Refine splitting Refine splitting Refine splitting