[Histonet] dapi and GFP problem problem
Andrea T. Hooper
anh2006 <@t> med.cornell.edu
Wed Jun 7 14:21:39 CDT 2006
I really do not think fixation is the problem. Speaking from
experience, for monolayers, aldehyde autofluorescence is RARELY a
problem. And without fixation your cells are not going to look so
great and in fact might look absolutely horrible!!!!
I feel fairly confident that the sealing of the Prolong Gold with
Permount did the damage.
>If you mount the unfixed cells do you have any special
>considerations? In other words do you have to view them right away,
>will the slide last, etc?
>On Jun 7, 2006, at 11:53 AM, Gayle Callis wrote:
>>You are probably experiencing autofluorescence induced the aldehyde
>>fixation. You can try Molecular Probes Image - IT image enhancer
>>that is supposed to get rid of the autofluorescence.
>>It is important to note that Prolong Gold antifade is supposed to
>>sit overnight and not sealed with fingernail polish, by curing
>>overnight, this mounting media, the refractive index gradually
>>increses as it cures. This may be why the control appeared black
>>just after you mounted the coverslip.
>>You can tell the CLSM to get rid of the autofluorescence too, some
>>might consider this cheating, but it can be done. What may be
>>better is to NOT fix the cells, and view them with CLSM - GFP is
>>designed for use with living cells and you may find the GFP is even
>>brighter. The Clontech manual called Living Colours tells you a
>>great deal about handling cells, etc with GFP. That manual can be
>>accessed online in pdf form.
>>Unfixed cells can also be mounted with Prolong Gold antifade w/
>>DAPI - we do this with unfixed frozen sections to avoid all
>>fixation or you can add a DAPI solution to the unfixed cells, and
>>view them without coverslipping or coverslipping with PBS.
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