Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

There is a third way (at least!) Li Liu.

Fix with an aldehyde based fixative and then permeablise with DMSO or a 
mild solvent like ethanol. This procedure is routinely used in 
neuroscience where you need to preserve the 3D structure of single 
neurons, for example. Compared with using a detergent, you get a lot 
less tissue distortion and damage: probably more of an issue in 
sections rather than cells growing on coverslips, but worth keeping in 
mind. Our experience is similar to tothers in that once the cells are 
just fixed, you can get some level of antibody penetration already...

IAN


On Friday, November 17, 2006, at 01:39  AM, Li Liu wrote:

> Search the CONFOCAL archive at 
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Greetings!
>  
> I have a question about how to fix cells:
> I need to label a plasma membrane protein with Alex488, and get images 
> with confocal microscopy, and I need the membrane to be permeablized. 
> I was told there are two ways to do it: either fix the cell with 2% 
> paraformaldehyde then permeablize the membrane with Triton; or fix the 
> cell with 100% methanol then don’t have to permeablize the membrane 
> since methanol will make holes on the membrane.
>  
> But for the best labeling of a membrane protein and get the best image 
> under the confocal microscopy, which one is better: fix cell with 
> paraformaldehyde or pure methanol?
>  


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Prof Ian Gibbins
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Flinders University
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