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Dear Florian,
Sometimes the Alexa dyes seem to form aggregates. I've mostly observed
this witrh the Alexa594 and less with the Alexa488. You can avoid the
sparklies by spinning either the stock solution or the final solution
at 10,000 rpm for 10 min. on a micro-centrifuge, then use only the
supernate.
regards,
Glen
On Mar 10, 2004, at 12:48 PM, Michael C. Adams wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> NO COMMERCIAL INTEREST
>
> Hi Florian. Could it be a fixation problem? What is your fix
> solution? Try
> making your fix solutions up fresh immediately before the experiment.
> This
> helps avoid weird fixation artifacts. Otherwise I would look to the
> antibody
> as the culprit. What is your source for the secondary? Not all
> antibodies
> are created equal: ergo, the reactivity can vary greatly between
> vendors. We
> find that Jackson Immunochemicals makes excellent secondary antibodies.
>
> Good Luck!
>
> Mike
>
>> ===== Original Message From Confocal Microscopy List
> <[log in to unmask]> =====
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> --
>> dear all,
>>
>> i am currently doing antibody stainings on paraffin sections.
>> unfortunately, i am always getting very bright dots when i look at
>> the staining under the confocal.when i do the control without first
>> antibody, i also see these dots. therefore, they should clearly come
>> from the 2nd antibody. it does not matter whether i use an alexa 488
>> or alexa 546 coupled antibody, they both produce these effects. maybe
>> the antibodies are bad, but they are from a commercial source and
>> work fine in many applications. so, is this a probelm related to
>> paraffin sectioning?
>>
>> i would appreciate any ideas or experience how to get around that
>> problem and be forever grateful!
>>
>>
>> best wishes,
>>
>> Florian
>> ----------------------------------------------------------------------
>> -------
> -----------------
>> Florian Ulrich
>> Heisenberg Lab
>> Max-Planck-Institute for Molecular Cell Biology and Genetics
>> Pfotenhauerstrasse 108
>> 01307 Dresden
>> Germany
>> phone: (+49) 351 210 2689
>> fax: (+49) 351 210 1489
>> email: [log in to unmask]
>
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
> Michael C. Adams
> Microscopy & Imaging Manager for Dr. Clare Waterman-Storer
> Laboratory of Cell Motility Studies
> Department of Cell Biology
> The Scripps Research Institute
> Attn: Mail Code CB-163
> 10550 North Torrey Pines Road
> La Jolla, CA 92037
>
> TEL 858.784.9244
> FAX 858.784.7521
> EMAIL [log in to unmask]
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
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
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