Hello Emma,
Try pre-labeling your primary antibody. This will eliminate the need to
apply a secondary antibody to the mouse tissue, without reducing the signal.
There are various methods for biotinylating your primary antibody and I have
included a manual from one of the iso IHC kits, which will give you a good
sense of what reagents are required and how the procedure works.
The basic concept:
1) incubate the primary antibody with a biotinylating reagent for 30
minutes, in a vial.
2) add a blocking reagent to that vial, incubate 3 minutes.
3) apply the labeled primary to the tissue.
4) apply streptavidin conjugate....etc. etc.
There are various reagents and systems on the market - I, ofcourse, endorse
the InnoGenex iso IHC kit. It's fast and consistent.
In any case, the procedure I have enclosed should be informative and inspire
you with a great many possibilities.
Good Luck,
Matthew Ogdie
InnoGenex
(925)543-1414
http://www.innogenex.com