[Histonet] Re: DAPI stained smeared nuclei
carl.hobbs <@t> kcl.ac.uk
Fri Aug 31 13:44:45 CDT 2012
When you use "smear", do you mean that the nuclei are blurring/ill-defined , with threads of DAPI-positivity coming away from the nuclei?
If so, it is due to underfixation.
Acetone is not a proper fixative.
In the time one uses is, it rapidly delipidises the tissue, rendering it better preserved, not fixed.
So, occassionally the nuclei "unravel".
A mixture of acetone: methanol overcomes this by coagulating nucleoproteins.
Sure, many times one does not notice significant "smearing" after acetone fixation.
No difference, in practise, between DAPI and Hoechst.
I use Hoechst: at a dilution factor of 1/20K it lasts quite a long time, lol.
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