[Histonet] immuno diluent vs. buffer question

[Michele Wich]

I have a question for those doing immunos by hand. I typically dilute my
primary, secondary and tertiary antibodies with manufacturer supplied
diluent. Recently, I was running a test to determine the source of a
mysterious precipitate that had begun to appear on my slides (It has
since then disappeared just as mysteriously). In one of the conditions,
I substituted the diluent with TBST for all dilutions. I found that this
particular slide had noticeably less background. When inquiring as to
what I should be diluting these reagents with, I received conflicting
answers from tech support. I heard that the secondary and tertiary
antibodies should be diluted with what they are actually in, i.e. .05%
TBS or 1M PBS. I also was told that I could dilute everything with
diluent. 

Can anyone tell me which is correct? Or why using the buffer yielded
less background than the diluent? Aside from sodium azide and BSA I
don't know what's in the proprietary reagent.


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