Is a negative residual stain value normal?

I have an H&E stained image. For the residual staining (stain 3), the red and green values are negative. Is this normal or do I need to re-estimate the staining vectors?

That is fairly normal in areas where another stain has high intensity. It will tend to become more severe as you have a higher Background set.

It happens with other stains too, for example you might see negative hematoxylin intensities where DAB staining is very dark or “perfectly DAB.”

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Thank you! I’m glad you’re here on this forum. You’ve been very, very helpful!

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