[Histonet] staining mouse tissues using mouse monoclonals

[alex.mclellan <@t> stonebow.otago.ac.nz]

Reply to Alan Meeker (10/28/03): staining mouse tissues using mouse monoclonals 

Dear Alan, 
The best fix will be to biotinylate your monoclonal (or purchase a biotinylated) and use 
labelled streptavidin to detect the signal. This would prevent reaction with Ig+ B cells in the 
tissue. 

However, if your reagent is too sparse and expensive to do an in-house biotinylation, I would 
perform the following slightly more complicated routine: First block the endogenous mouse 
Ig on the tissue with a Jackson anti-mouse Ig Fab Ab (donkey or goat would be cleanest) 
and wash away excess. A Fab Ab is the key here to avoid later reaction against your 
monoclonal, as would happen with a divalent Ig (eg. whole or Fab)2 molecule. Then react 
the section with your monoclonal and use a labelled anti-mouse Ab to detect. I have had a 
lot of luck using Jackson Ab (preferably donkey), one as Fab form (to block) and the other as 
whole or (Fab)2 to detect (labelled with HRP). The detection could also be, but need not, be 
a Fab form. The specificities and affinities of the blocker and detection Ab (against mouse 
Ig) should be as similar as possible for obvious reasons, so try to choose from the same 
company (who may also be able to advise on the best choice). 
--
A.D. McLellan
Department of Microbiology
University of Otago
PO Box 56
Dunedin
New Zealand
Tel: +64 3 479 7728, Lab: +64 3 479 7147
Fax: +64 3 479 8540
http://microbes.otago.ac.nz/dept/index.html