[Histonet] Problem wih FITC secondary "unbinding" after about 10 hours (using immunofluorescence)

[Janelle Pakan]

We are having a very strange problem with our FITC-conjugated secondary in
double labeling immunofluorescence processing. We are processing for 3
different enzymes using a Rhodamine secondary and these seem to be fine
throughout the process, but using 3 different markers (GFAP, MAP2, OX42)
with a FITC conjugated secondary we see something very strange. The labeling
looks really nice immediately after processing and mounting for up to 6-10
hours later, then when left in the fridge overnight, the next day the FITC
(and only the FITC, the rhodamine is fine) seems to "unbind" and is all over
the tissue creating a "background" type fluorescence.

I have tried 4 different secondaries from 3 different companies (although
they are all FITC conjugated - want to try a cy2 or alexafluor 488 but don't
have any yet) and they all do they same thing - really nice immediately
after processing, but cell labeling is "gone" by the next day and appears as
specks all over the tissue sample.

We are using 4%PFA fixed, floating rat brain sections (used three different
brains so far and all had the same effect).

These are the other things I checked:
- used 3 different mounting media (Fluoromount-G, Elvanol, 90% glycerol):
all had same effect. Leaving tissue overnight in PBS in fridge also had same
effect, so figure it cant be anything with the mounting process.
- used 3 different primaries and all have same effect
- we use ovoid (BR0014G) PBS tablets (1 per 100ml, pH 7.3) - commercially
available standard PBS tablets... should be okay?? Anyone else use this?

Any help would be appreciated, or just to know if anyone has seen this
before. I did immuno for 5 years in a different lab and never saw anything
like this, but recently switched labs and had this problem immediately. I
figure it must be something in the solutions I am using n the new lab, but I
just cant fathom what it would be at this point.

Thanks

Dr. Janelle Pakan
University of British Columbia
Brain Research Centre
2211 Westbrook Mall
Vancouver, BC
Canada