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

Courses for horses, again.  At the LM level, a freeze-thaw treated  
vibratome section retains much better morphology than a cryostat  
section, often with improved immuno-labeling.  At the EM level, its   
just ground meat.

Regards,
Glen


On Nov 19, 2006, at 2:13 PM, ian gibbins wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Surprisingly good for light microscopy, Glen, but you should see it  
> at TEM level !
>
> This freezing procedure can generate massive disruption of many of  
> the intracellular organelles (eg mitochondria, ER) and can also  
> cause significant translocation of cytoplasm, presumably due to  
> large ice crystal formation. if you are lucky, you get big  
> fractures in the cells rather than microdamage, but either way, you  
> wouldn't want to be doing high resolution subcellular localisation  
> of freeze-thawed tissue.
>
> hope that helps
>
> IAN
>
>
>
> On Saturday, November 18, 2006, at 03:23  AM, Glen MacDonald wrote:
>
>> 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]
>>
>> ********************************************************************* 
>> *********
>> The box said "Requires Windows 95 or better", so I bought a  
>> Macintosh.
>> ********************************************************************* 
>> *********
>>
>>
>> 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> .
>>>
>>
>>
>
> * * * * * * * * * * *
> Prof Ian Gibbins
> Anatomy & Histology
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