Re: [Histonet] re: Some details about my questions on mouse olfactory

From:Gayle Callis

A few more thoughts.

One way to get rid of unwanted fluorescent aggregates is to spin down a 
diluted antibody-fluorophore just before application.  We call this 
annoying problem "glowing garbage" although most of the time it is not fine 
punctate dots, but bigger clumps of stuff.

Also, if one uses Strepavidin-Alexa dye conjugates and the tissue is 
notorious (or you want to just make sure) for containing endogenous biotin, 
a Strepavidin or avidin biotin block step should be added.  Vector is one 
source for both kits.

Molecular Probes advised never diluting Strepavidin-Alexa conjugates with 
normal serum in the buffer, the SA can bind to any endogenous biotin found 
in the normal serum.   A buffer containing Tween 20 was acceptable.

At 11:25 AM 4/13/2006, you wrote:
>Just to confirm: do you get the same "endog. pattern" when using DAB-based
>immunolocalisation? If not, and they are normal tissues, one should not get
>those "unspecific dots", UNLESS they are due to aggregates of the
>fluorochrome-labelled secondary reagent.
>A pic would be good.
>  Try posting here  as well?
>Also, are you using free-floating sections?
>Depending on the requirements, I will take a perfused-fixed brain and and
>cut it parasaggitally.Then further fix, as you do. However, if I'm
>interested in the OB only, I'll cut them off after disection and fix
>separately. Given that your procedure is as you state, I can't see a problem
>with "inadequate " fixation.
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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