[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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