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Dear Pedro-
The antibody will not cross the cell membrane before fixation.  Cells
are impermeable except to certain very low molecular weight or
hydrophobic compounds. Even formaldehyde fixation does not open up
big enough pores to get an antibody through, as the antibody is very
big.  If you are seeing staining after stretching the muscle strips,
it is probably because you are breaking the cells and then the Ab can
penetrate.  If you fix in a removable medium, such as Carbowax, you
can make 1-2 micron sections and do the staining on them.
Carol

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear Carol
>
>no, our samples are isolated cells (dispersed using papaine/collagenase
>digestion) or strips of whole, intact gallbladder smooth muscle. We treat
>the sample and then we fix with paraformald.
>
>At the end of the day, it could be that our result is correct: we´re
>seeing now some nuclear signal for creb in intact muscle when we stretch
>the strips during the fixation protocol (stretching is usual when handling
>smooth muscle strips for contraction/fixation/dispersion techniques). This
>signal decreases upon stimulation with different stimuli. Perhaps the
>problem is simply low signal or low efficiency of the antibody in our
>samples.
>
>Thanks for your reply
>
>
>Carol Heckman wrote
>
>>  Dear Pedro-
>>  It is an interesting problem.  Are you working on frozen sections of
>>  exocrine cells?  If so, at least once in a while the nucleus must be
>>  cut in half and the Ab must penetrate to the site where the CREB is
>>  supposed to be.
>>  carol
>>
>>
>>>Search the CONFOCAL archive at
>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>>Hi all,
>>>
>>>not strictly confocal question
>>>
>>>We´re using antibodies to determine CREB phosphorylation and
>>>  translocation
>>>of NFAT to nuclei of glandular (exocrine pancreas) and smooth muscle
>>>cells. After several weeks changing Ab dilution, incubation time, block
>>>solution,...we can´t see in the nuclei a decent signal of phosphorylated
>>>CREB (supposed the only place for this molecule) or NFAT. We suspect that
>>>we are simply failing to detect the presence of labelled NFAT/CREB in the
>>>nuclei (NFAT in cytosol decreases when we apply stimuli known to induce
>>>nuclear accumulation)
>>>Is it possible that some cell types have a more "impermeant" nuclear
>>>membrane? We have already prolongued the incubation time in triton.
>>>Any idea would be wellcome
>>>
>
>
>--
>Dr Pedro J Camello
>Dpt Physiology
>Faculty of Veterinary Sciences
>University of Extremadura
>10071 Caceres
>Spain
>Ph: 927257100 Extension 1321
>Fax:927257110

--
__
Carol A. Heckman, Ph.D.
Professor of Biological Sciences
Director, Center for Microscopy & Microanalysis
Bowling Green State University, Bowling Green, OH 43403
fax: (419) 372-2024       email: [log in to unmask]
http://www.bgsu.edu/departments/biology/facilities/MnM
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