Immunofluorescence - (Apr/04/2010 )
does anyone have experience with double-immunofluorescent staining with primary antibodies raised IN THE SAME SPECIES!?
Would be very grateful for useful tips how to handle this and whether it is at all advisable.
I have tried staining two rabbit antibodies on the same section using the Zenon kit. It worked ok, although I was never really sure if the staining was indeed specific. After a time you'll lose the signal, so better hurry up. I'd rather recommend using Ab from different species, if possible.
So long as you use the primaries sequentially (i.e. go through the proceedure with one, including secondary and then go back and repeat with the other), and ensure that the 2ndary steps go to saturation (you will need to play with this a bit), you shouldn't have a problem. It would also be a good idea to have a slide or two stained with only one primary and its respective secondary.
bob1 on Apr 7 2010, 01:34 AM said:
We used the same protocol, worked well once we had it optomised but optomising did take a bit of time. It makes a difference which primary/secondary combo you use first so try both ways, and as Bob1 said, make sure the secondary antibody steps are saturating or you'll be in a world of frustration!