[Histonet] Directly conjugated secondaries versus biotinylated secondaries

[Adam .]

Greetings,

Hopefully all of you are enjoying a great time with your friends and family
this week rather than working like me. Here is my problem.

I have an antibody (anti-panendothelial antigen, i.e. MECA32 from BD
http://www.bdbiosciences.com/ptProduct.jsp?prodId=19506) and I'm trying to
get it to work on 4% PFA fixed, decalcified, paraffin embedded sections. I
previously titered it to 2 ug / mL with a biotinylated anti-rat F(ab')2 (1
ug / mL) and then a strepavidin Dylight 594 (1 ug / mL) and got beautiful,
strong staining. Those of you who have been helping me from my IHC infancy
would be proud.

However, I really need it to be a directly conjugated secondary because I
want to co-stain with something else that only works with a biotinylated
secondary followed by tyramide amplification. So I retitered it from 5 ug /
mL and down and came in with a Dylight-649 conjugated anti-rat (1 ug / mL)
secondary and I "saw" nothing (Dylight 649, which is a Cy5 replacement, is
too far red to see without the aid of a camera--although the guy who helped
me use the microscope says there's one woman on campus who has infrared
vision). I should note that another group has reported getting this to work
with a biotinylated primary and adding an fluorophore conjugated avidin.

I can think of a couple of possibilities here
1) The antibody needs that extra edge of a biotinylation to get sufficient
signal (has anyone ever seen this problem which couldn't be solved by
titrating something?)
2) I need to increase the primary titer ever more
3) I need to increase the secondary titer (what primary titer should I use
if I do this?)
4) Dylight 649 is just too dim to see anything and if I switched to another
fluor, it would work. I have no experience with this fluor, as our floor
scope doesn't have the filters for it.
5) This is all a fluke and I screwed something up

Any suggestions on how to troubleshoot this are welcome.

Adam