Help with DAB Analysis app please
Hello everyone.
I have started using Image-Pro Premier to analyse images of mouse artery specimens that have undergone immunohistochemical staining using DAB. I have some questions related to my DAB analysis. I have read and watched the Media Cybernetics information on the DAB analysis app (including video), and have read many posts online, but I still have unanswered questions.
(1) I find that the larger the region of interest, the higher the mean density. Why is this the case? I expect that the selected area would be greater, but the mean density doesn't make sense. For example, if I have a specimen that has two arteries next to each other, and I perform a DAB analysis, the mean density will be much, much higher than if I cropped one cross section out and just analysed one artery.
(2) I find that specimens with a visibly high amount of staining (lots of dark brown staining everywhere) seem to have low mean densities compared to those with visibly less staining. I assumed that a higher mean density implied more staining. Is it actually the opposite?
(3) Since IOD = area x mean density, I don't understand how IOD is able to validly compare DAB staining between different tissue specimens with different-sized regions of interest. Hence, mean density seems to be the way to go in order to compare staining between different specimens, but my findings don't make logical sense to me.
Can someone please explain how to interpret the output from the DAB analysis app?
Thank you very much,
Margaret.
I have started using Image-Pro Premier to analyse images of mouse artery specimens that have undergone immunohistochemical staining using DAB. I have some questions related to my DAB analysis. I have read and watched the Media Cybernetics information on the DAB analysis app (including video), and have read many posts online, but I still have unanswered questions.
(1) I find that the larger the region of interest, the higher the mean density. Why is this the case? I expect that the selected area would be greater, but the mean density doesn't make sense. For example, if I have a specimen that has two arteries next to each other, and I perform a DAB analysis, the mean density will be much, much higher than if I cropped one cross section out and just analysed one artery.
(2) I find that specimens with a visibly high amount of staining (lots of dark brown staining everywhere) seem to have low mean densities compared to those with visibly less staining. I assumed that a higher mean density implied more staining. Is it actually the opposite?
(3) Since IOD = area x mean density, I don't understand how IOD is able to validly compare DAB staining between different tissue specimens with different-sized regions of interest. Hence, mean density seems to be the way to go in order to compare staining between different specimens, but my findings don't make logical sense to me.
Can someone please explain how to interpret the output from the DAB analysis app?
Thank you very much,
Margaret.
0
Answers
Your concerns are related and doesn't make sense in the way you described these issues, but could you please post some examples. I suspect, the intensity calibration may be involved in this as well.
Thank you,
Nikita.