alexkoon

I am trying to do double staining with two rabbit primary antibodies in a sequential way. Let's call them rb anti-alpha and rb anti-beta. I set up 4 conditions:
(1) No-rb anti-alpha --> anti-rb Alexa488 --> 4% para fix 5 min --> No rb anti-beta --> anti-rb Alexa594
(2) No-rb anti-alpha --> anti-rb Alexa488 --> 4% para fix 5 min --> rb anti-beta --> anti-rb Alexa594
(3) rb anti-alpha --> anti-rb Alexa488 --> 4% para fix 5 min --> No rb anti-beta --> anti-rb Alexa594
(4) rb anti-alpha --> anti-rb Alexa488 --> 4% para fix 5 min --> rb anti-beta --> anti-rb Alexa594

In a sequential staining like this, the most worry comes from the 2nd secondary antibody (594 here) binding to the 1st primary antibody (anti-alpha here). So the fixing in the middle is a measure to prevent this.

The result of this expt was as follow:
(1) No signals in 488 and 594
(2) No signals in 488 and 594
(3) Signals in 488, but not 594
(4) Signals in 488 and 594

I was expecting signals in 594 for condition 2, but I did not get it. I thought it was a technical error, but the same result was repeated 3 times. Since there's no 594 signals in condition 3, there should not be any significant binding of anti-rb 594 to anti-alpha. So that would mean the 594 signal in condition 4 is real. But without anti-alpha, I did not get the 594 signal in condition 2. So I'm very puzzled. Can someone please help? Thanks!