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

The is also evidence that Triton can extract the proteins, as well.   
Having said that, soaking the cells or slice in SDS, 0.25% to 1% for  
10 min.  can greatly increase labeling with some antibodies.

Another trick for thicker samples, like 40 µm vibratome slices, move  
the sample through graded sucrose to 30% sucrose, freeze at about -80  
C, then thaw in a refrigerator.  If your goal is to punch holes in  
the membranes, this will do it.  the morphology is surprisingly good.

Regards,
Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
(206) 616-4156
[log in to unmask]

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On Nov 16, 2006, at 6:49 PM, Stephen Bunnell wrote:

> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- 
> bin/wa?S1=confocal For several GFP-tagged integral membrane  
> proteins and raft proteins we have observed that Triton extraction  
> induces ‘clumps’ that are never observed in the live cells.
>
> For several of these proteins, antigenicity was also dramatically  
> reduced by fixation in 3.7% paraformaldehyde.
>
> We have often had very good success with greatly reduced doses of  
> paraformaldehyde; doses as low as 0.4% will often give you complete  
> fixation with better epitope preservation. Very low doses of  
> saponin can be used instead of Triton; the extraction of the  
> membrane seems far less severe. A mild alcohol extraction was also  
> mentioned... We may try this ourselves.
>
> Of course, these suggestions are just meant to provide a starting  
> point... I expect that you will need to titrate your conditions to  
> achieve the best possible results.
>
>     Best regards,
>
>
> Stephen C. Bunnell, Ph.D.
> Assistant Professor
> Tufts University Medical School
> Department of Pathology
> Jaharis Bldg., Room 512
> 150 Harrison Ave.
> Boston, MA 02111
>
> Phone: (617) 636-2174
> Fax:   (617) 636-2990
> Email: [log in to unmask]
>
>
> On 11/16/06 10:09 AM, "Li Liu" <[log in to unmask]> 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?
>>
>>
>>
>> Thanks!
>>
>>
>>
>> Li
>>
>>
>>
>>
>> ----------
>> Li Liu
>> Molecular and Cellular Physiology
>> University of Cincinnati, College of Medicine
>> Cincinnati, OH 45267-0576
>> Email: [log in to unmask]
>>
>>
>> Everyone is raving about the all-new Yahoo! Mail beta <http:// 
>> us.rd.yahoo.com/evt=42297/*http://advision.webevents.yahoo.com/ 
>> mailbeta> .
>