I'm taking over a project where all the primary ab have already been added to the slides. The person before me didn't document his work very well, I know he used donkey serum before adding the primary ab and then some of them he used goat serum. No idea which ones.
So I'm wondering if it makes a big difference if I used a anti-mouse IgG (secondary Ab) raised in goat if he incubated the primary ab with donkey serum since the rule of thumb is to use the same species as ur secondary ab...I can't find any literature on using serum and ab of different species. (primary ab was raised in mouse and it's on a mouse tissue).
Thanks
Hi, although I can't be of any specific help regarding the donkey/goat question, there is an extremely interesting paper that suggests that serum blocking really has no effect on IHC at all (or even a detrimental effect). With this in mind, perhaps it would be OK to proceed with the goat anti-mouse without real worries.
Link to paper below:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3216515/pdf/srep00028.pdf
it really doesnt matter which serum you use. many poeple just use BSA for blocking and proceed with antibodies. I tried both BSA and secondary ab specific serum and there was literally no visual difference. And I also agree with Sarah Mason, above mentioned paper showed blocking is not necessary at all. you can simply go on with your secondary antibody. Good Luck.
There are no relation between serum and primary antibodies species. You can use BSA, milk, or any specie serum. I work with cattle, and usually primary antibodies is mouse specific, then I need to use blocking to decrease background.
Our laboratory has (inadvertently) done exactly what you describe - that is, block in donkey and use goat secondaries. We also were not able to tell a difference between this and a round of staining that used the same species for both. I don't think there is much to worry about, but for sanity purposes it may be nice to use the same species moving forward.