Hi, I am trying to incubate a rat pancreatic section with two different antibodies (co-incubation):
1. Anti pancreatic polypeptide (goat polyclonal) - did a solo staining and works on rat's pancreas
2. Anti TTMP (rabbit polyclonal) - supposed to work on rats
The secondary antibodies that I am using are:
1. Rabbit anti-goat (ab50623) - for anti pp
2. Goat anti-rabbit (ab6717) - for anti ttmp
I have done staining on rat's pancreas section using anti-pp + the rabbit anti-goat secondary and it worked fine. But when I mixed both antibodies (anti pp & anti ttmp), I couldn't get any staining for both antibodies.
I also tried incubating the primaries separately. I incubated the slide with anti pp first then washed with PBS 3X then I incubated the slide with anti TTMP. Still no staining observed.
I believe it has something to do with the secondary, maybe? Because if you take a look at the secondaries that I am using, they were raised in the same animal that the other one's anti to. If that is the problem then what should I use? Should I buy a completely different secondaries that have no connection to either rabbit or goat? I browsed the list of antibodies but don't see any other animals aside from rabbit, goat and donkey!
i agree it might be the secondary, have you tried using donkey anti-goat and donkey anti-rabbit? and block with donkey serum if you are using serum. Donkey anti-goat and donkey anti-rabbit secondaries can be found by lifetechnologies (thermofisher)
The rule of thumb is to get a secondary Ab that was raised against the species from which the primary antibody was derived, which was executed here.
However, since you are using polyclonal and not monoclonal antibodies, there is a tendency for the antibodies to cross react in one way or the other. So you may consider processing with each Ab separately.
Alternatively, you may also try to get Anti TTMP; goat polyclonal and then retry the combined or split incubation periods together with the Anti pancreatic polypeptide as you did earlier.
Let me know how it turns out.
Hi Elena, You are right the reason for your negativ stain result are the scondaries.
If you use this two secondary ab in a cocktail the rb-A-gt bind to gt-A-rb and the other way round. So when you put this cocktail on your sections ther are not enough free binding sides for the primary ABs. I recommend either to use secondaries generated in the same species like Stephen told you or replace the rb-A-gt for example to Horse-Anti Goat. It works as well. Good luck!
Hi all, thanks for your replies.
Unfortunately I cannot get TTMP raised in any other animals since my project predecessor had raised this antibody in rabbits (so I have to make use of whatever resources available).
I can however buy new secondaries or better yet I'll borrow some from other labs first and see if it works.
Thanks again everyone. Will let you guys know the outcome.
i tried co-staining of hepatic stellate cells with smooth muscle actin and anti-TNC antibodies. after finishing the anti-TNC in incubated the slides in Hcl 0.01 N for 10 min , then washed with PBS then blocking with 10% normal goat serum followed by incubation with the 2nd antibody
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