H-DAB and Quantifying protein expression

Hey guys,

Am I right in saying that DAB stains for intracellular (but not nuclear) proteins and Haeamtoxylin only stains for nuclear proteins only. I am analysing cytosolic proteins (PLOD2 and NICD) and RhoE (which can shuttle between the nucleus and cytosol). I’m a novice to QuPath and I’m thinking that the best way to analyse the expression of these proteins would be to create a threshold for DAB and Haematoxylin and then run positive cell detection?

Am I right in saying that ‘positive cell detection’ just includes cells that meet the DAB/Haematoxylin threshold?

Any help would be much appreciated!

Thank you

DAB can be used for either cytoplasmic or nuclear markers, it really depends on your target antigen (primary antibody). Depending how much of the cell was captured in the cross section it may appear to have some overlap from one compartment to the other. Here is some useful information about chromogenic IHC Intro to Immunohistochemistry (IHC) Staining: Steps & Best Practices : Leica Biosystems

Hematoxylin binds to DNA & RNA, hence it is safe to assume it is nuclear.

It will be easier to help you find suitable method If you can post a sample image.

In QuPath cell detection, Hematoxylin threshold is simply used to find, identify and segment nuclei. Then an artificial cytoplasm compartment is created around the nuclei by expanding it with the size you specify in the cell detection parameters.

Assuming the color deconvolution worked well, you can set a threshold for DAB intensity in the positive cell detection. You can choose which compartment (nuclei, cytoplasm or whole cell) to evaluate for DAB intensity. Cells with DAB intensity above the threshold value will be classified as ‘Positive’. Hematoxylin threshold is not used for positivity.

Hope this information helps.

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Hi Ajay, that is extremely helpful, thanks very much!

I’ve attached some images below - these are tissue sections of the skin. Essentially, what my team and I are hoping to analyse is the number of dermal fibroblasts /sq micron (proliferation analysis) and the expression of a protein (RhoE) that is found in fibroblasts and can shuttle between the nucleus and cytoplasm.

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How are you planning to identify the dermal fibroblasts ? In your Images what is the DAB used for, proliferation marker or the RhoE ? If you want to quantify expression of a protein you may want to use fluorescence instead of chromogenic IHC. It will also allow you to have multiple markers that should help with identification of correct cells (phenotype) I would look at this thread for relevant discussion: Mesure color intensity of DAB image
And this post, Mesure color intensity of DAB image - #4 by gabriel
someone else may have better suggestion since I don’t have much experience with your specific assay.

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