I'm using LTP-induced rat brain slice for IHC
and I want to check BrdU, PCNA, GFAP and DCX
So, I did double labelling like this.
1. BrdU/PCNA
2. BrdU/GFAP
3. BrdU/DCX
4. PCNA/DCX
5. PCNA/GFAP
Hoechst is added to all of these.
But,, only first and second combinations were stained.
moreover, I couldn't see any nucleus(Hoechst) at all!

so, I checked whether each of antibodies(for BrdU, PCNA,
DCX and GFAP) work using tissues of the same brain.
and it did and I could see nucleus, too.

then, I did those double labelling thing again.
and the result was same with first trial.
Hmmm, do they interfere each other?
I don't know what the problem is!
Do you have any suggestion?

So in your first two stainings, did you see both were stained or only one of them was?

Both were stained.
Brdu, PCNA and GFAP were all good though hoest was gone...