[Histonet] Re: Sheep anti BrdU

[Johnson, Teri]

Yun,

Do you currently have an anti-BrdU made in mouse that is working for
you? And are you using chromogenic or fluorescent techniques?  If you
are using chromogenic, consider biotinylating one of your mouse
monoclonals to do the double staining. Do a sequential immunostain
technique using your 1st mouse antibody, anti-mouse secondary, DAB (or
other stable) chromogen, then do the biotinylated antibody,
ABC/streptavidin or avidin biotin/LSAB Alk Phos label, and chromogen of
choice after that.

If you are using fluorescent techniques, it might be best to use the
sheep anti-BrdU in a cocktail and do your double staining that way.

Another thing to consider when doing BrdU doublestaining is what effect
the denaturing step will have on the antigenicity of your other proteins
of interest.  We had some challenges trying to do BrdU doublestaining
because of this.  Most often when working up antibodies, you don't
usually include this step.  Therefore trying to just plug in an antibody
that has previously worked ok in the single staining techniques may or
may not work well when combined with the protocols needed for BrdU
staining.

Good luck, and let us know how the sheep antibody works if you decide to
go that route.

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133