Hi there,
I have two targets I want to dual label for immunofluorescence. One ab is raised in sheep and other in rabbit. So I need an anti-sheep and anti-rabbit secondary alexafluor.
Now I understand that from most sources online, it is best to use secondary antibodies raised in the same species as your blocking agent, i.e. block in goat serum, use a goat anti-xx secondary antibody.
However, I've been told by postdoc that if I do this, then its likely that the secondary abs will bind to the background block, resulting in significant background fluorescence. He said I should block in a serum that is essentially neutral to my experiment, such as bovine (even though I probably can't use bovine serum because of the IgG cross-reaction between sheep and cow).
So now I'm thinking I should use Goat serum to block and use Donkey anti-sheep/anti-rabbit alexafluors to dual label.
Could someone please offer me some advice on this?
Thanks
Hello David,
I don't think it is vital, they recommend it but it is not compulsory, as long as you have your proper positive and negative controls.
I agree with your postdoc's advice, I had a certain goat-antibody and when I blocked with goat serum I had a lot of non-specific binding (+ high background) in both IF and IHC. So, for that antibody I used horse serum instead (on the sample, +ve and -ve controls).
I believe what you propose is OK. I've been using goat serum to block and donkey anti-bodies and it gave good results.
Best and good luck,
Ahmed
Like Ahmed said, goat serum to block and anti-donkey secondary should work ok. However, should you need an alternative you can try using fish or porcine skin gelatin to block as well. You can read more about it here-https://biotium.com/product/fish-gelatin-blocking-buffer-10x/. I have used one from sigma aldrich in the past for mouse epidermis whole mount immunofluorescence with success.
Good luck!
Vagisha
https://biotium.com/product/fish-gelatin-blocking-buffer-10x/
I never use serum as blocking agent in my IF, instead I use BSA to 4 to 2%. Is your sample a piece of tissue or a layer of cells?
Hi,
I think it's best as everybody said.
A more important point is to know if your protein of interest is present or not in serum because that would affect the efficiency of the antibody for the staining (I know what I am talking about :( )
Therefore, I would replace by a solution of 3%BSA (in TBS or PBS)
Hope that helped
S
Samples are brain tissue from quail
I'm going to try donkey serum initially and then see how it is. Other researchers in our school have blocked in serum where the species is the same as the secondary and their brain sections appear fine....albeit mammalian brain. I have plenty sections to validate everything.
Thanks for all your help.
The reason is to minimize non-specific binding. Try using polyvinylpyrrolidone (PVP), it is a non-protein blocking agent. I have successfully used 0.25% for blocking and you can dilute both your primary and secondary in it.
No, it is not at all necessary to use blocking serum as in my case I have mainly used 1%BSA in blocking buffer and it has worked fine.
Use 2nd antibody raised from teo different species…it will reduce the cross binding of target antigens.
All d best!
Yes, It is very important to use secondary antibodies with different spices like goat and another mouse (mice) for the prevention of cross binding of targeted antigens.
- Badges
- Science method