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If you use a different secondary (doesn't really matter what kind) on 
the same samples, do you get the punctate effect?  Do you know how 
much protein you are using in the Alexa secondary (maybe it is rather 
high)?

None of those questions solve your problem of course.  If you trust 
your primary, why not label it and go direct?  We have had great 
success with Alexa-lableled primaries.  The preparation is simple and 
takes less than one day.  Down side is that you need min 0.5 mg of 
antibody to label, and that one really ought to double the 
recommended reactive Alexa concentration to get good labeling the 
first time around.  If you're a cheapskate, you can do it with the 
recommended concentration and, if you want hotter product, then just 
repeat it.  A final caveat - we use polyclonals "your results with 
monoclonals may differ"......

Rob Palmer

>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
>***************************************************************************


-- 
Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-594-0025
fax 301-402-0396