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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]

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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
>
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> * Tina (Weatherby) Carvalho               * [log in to unmask]
> * Biological Electron Microscope Facility * (808) 956-6251
> * University of Hawaii at Manoa           * http:// 
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