[Histonet] Double immunostaining

Thanks all for the very considered answers to my last question.

1. I would like to enquire - I am planning on using rhodamine and FITC to double stain my tissue slides with anti-p21 and anti-p27. Unfortunately, both are nuclear=20 proteins, and I have heard horror stories from a couple of colleagues on the difficulties of staining the same structure with different dyes. According to them, usually only one dye is predominant. Does anyone have any experiences attempting to stain the nucleus with two different dyes, or would the recommended approach be to stain two adjacent sections?

2. I have noted an apparent artefact during my ABC-DAB immunohistochemical staining of tissue. The tissue border stains heavily (an obvious artefact), but just below the border, there is a rim which completely doesn't stain at all, in stark contrast to the centre of the section. Has anyone else experienced this?

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