threshold of DAB image

[Sarah Mohamed]

Hi,
I would like to ask you about threshold method for determining area of brown DAB-stained cells.
i found 2 methods and each gives a different area fraction (84%, 64% ). can`t determine which one to use!first one as in this link by Sean Lauber (openwetware.org/wiki/Sean_Lauber:ImageJ_-_Threshold_Analysis)
 and other one is by changing the image type to greyscale 8 bits and then choose image-adjust-threshold -auto-apply. then mark dark background & then clt+M for brown area fraction measurement (and in set measurement display label is checked)
Also,
Should the area value be the same if using threshold method or color deconvolution?Thanks in advance.Regards,
Sarah M. Mosaad, M.Sc.


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