Hi I have treated my cells with an agent that causes my fibroblasts 3t3 to rapidly multiply. When I counted my cells, I found that the cells are almost double of control. I wanted to validate proliferation with ki67, and found that the treated cells do not have the characteristic nuclear expression but rather the whole cell showed fluorescence. It couldn't have been staining error as I did the control together and that came out ideal nuclear expression (see attached). Initially I thought it could be autofluorescence, in that case my treated cells are Ki67 negative, which is bizzare given how proliferative they are. When I visualised the treated cells at a higher magnification, I saw distinct dotted structures (punktas) all across the cells in treated cells (see 2nd image). This suggests that it could not have been autofluorescence, as autofluorescence looks pretty dull and smooth. I have read papers where severe grade cancers having cytoplasmic and membrane expression of Ki67. Can anyone help me out with the interpretation? Any experiences or comments on this? (Sorry for the bad quality of images as I had to click it from my laptop screen).
PS I have replicated the experiment thrice.
PS 1. All images of control and test were taken from epifluorescence microscope at the same exposure time.
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