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I concur with Glen's assessment. In trying surface labelling of cultured
cells, we had an antibody against a receptor known to be on the cell
surface. Live staining showed nothing. After fixation, the cells lit up
clearly. Our conclusion: Fixation permeablized the cells, and the antibody
was to an epitope on the receptor on the inside of the membrane. The live
cell staining protocol for another receptor (5 min, diluted in culture
medium without serum) worked well .
Good luck,
carl
Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX 520-621-3709
----- Original Message -----
From: "Glen MacDonald" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Friday, March 31, 2006 3:18 PM
Subject: Re: Background or precipitate with AlexaFluor 488
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Tina,
> Does this punctate label appear as pinpoints of light? I've never
> observed this with Alexa 488 but Alexa594 seems prone to aggregation
> which covers the sample with tiny pinpoints of light. Try centrifuging
> the secondary, 10,000 rpm for 10 min. and apply the supernate.
>
>
> As far as that other question posted on background, there is simply no
> fixative that will not permeabilize the membranes. Coagulating
> fixatives such as methanol or acetone remove the lipids that bar entry of
> the antibodies, and are often used post-fixation to increase penetration
> following aldehyde fixation.
>
> The internal label may be receptor protein or receptor subunits
> synthesized but not yet inserted in the membrane.
>
> 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 Mar 31, 2006, at 1:23 PM, Tina Carvalho wrote:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hi, All-
>>
>> I am having an intermittant problem with a strong, punctate
>> background/nonspecific labeling or precipitate with AlexaFluor 488 in
>> two
>> very different applications. One project involves flounder sperm that
>> I've
>> fixed and put onto poly-L-lysine or gelatin coated slides, rinsed,
>> blocked, primary antibody for various lengths of times, rinsed, then
>> applied secondary ab with AlexaFluor. The other is sections of LR
>> White-embedded Arabidosis leaves, treated roughly as above. Doesn't
>> happen
>> all the time, or even on all slides of a single run, and I haven't been
>> able to trouble-shoot this at all.
>>
>> It looks more like a precipitate than nonspecific labeling, appears on
>> both the slides and the sperm or LR White sections. This has been only
>> annoying so far, but my next project with the sperm is to try to label
>> membrane receptors, so bright, punctate green Christmas ornaments just
>> will not do!
>>
>> I had a similar problem when looking for IL10 receptors in cultured
>> fibroblasts, and the AlexaFluor 568 ornamented everything; standing up
>> tall enough to sway in the mounting medium. In that case I think it was
>> because the primary antibody had been made in BSA, which we hadn't
>> known,
>> and we were blocking with BSA... (And if any of you has had success with
>> looking for cell surface receptors, I'd like to hear from you!).
>>
>> Any ideas? I'd appreciate any insights!
>>
>> Mahalo,
>> Tina
>>
>> **********************************************************************
>> *****
>> * Tina (Weatherby) Carvalho * [log in to unmask]
>> * Biological Electron Microscope Facility * (808) 956-6251
>> * University of Hawaii at Manoa * http://
>> www.pbrc.hawaii.edu/bemf
>> **********************************************************************
>> *****
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