Blocking in Immunofluorescence Microscopy - (Jun/21/2007 )
Hi everyone,
I have 2 questions about immunofluorescence microscopy preparations. In most of the protocols it is recommended to use the serum of the secondory antibody species. If the secondary antibody is goat antimouse, we should use the goat serum for blocking. But recently some one told me that they always use the serum of a different species. For example, if the secondary antibody is goat antimouse, they use donkey serum for blocking. It was very weird for me. Which method are you using and why?
And the second question: Do you rinse your samples after blocking and before primary antibody incubation? Some protocols suggest not to rinse the samples between these two steps whereas others rinse 2-3 times before adding the primary antibody. Which method you use?
Thanks a lot for the help
I have 2 questions about immunofluorescence microscopy preparations. In most of the protocols it is recommended to use the serum of the secondory antibody species. If the secondary antibody is goat antimouse, we should use the goat serum for blocking. But recently some one told me that they always use the serum of a different species. For example, if the secondary antibody is goat antimouse, they use donkey serum for blocking. It was very weird for me. Which method are you using and why?
And the second question: Do you rinse your samples after blocking and before primary antibody incubation? Some protocols suggest not to rinse the samples between these two steps whereas others rinse 2-3 times before adding the primary antibody. Which method you use?
Thanks a lot for the help
We use NGS for blocking when using secondary antibodies made in goat. I don't think using a donkey serum for blocking and then using goat secondaries would be a problem. To answer your second question, I don't wash in between the primary antibody and blocking steps.
I have 2 questions about immunofluorescence microscopy preparations. In most of the protocols it is recommended to use the serum of the secondory antibody species. If the secondary antibody is goat antimouse, we should use the goat serum for blocking. But recently some one told me that they always use the serum of a different species. For example, if the secondary antibody is goat antimouse, they use donkey serum for blocking. It was very weird for me. Which method are you using and why?
And the second question: Do you rinse your samples after blocking and before primary antibody incubation? Some protocols suggest not to rinse the samples between these two steps whereas others rinse 2-3 times before adding the primary antibody. Which method you use?
Thanks a lot for the help
This is from Jackson Product Catalog, technical information: "Never block with normal serum or IgG from the host species of the primary antibody when a labeled secondary antibody is used. If immunoglobulins in the serum bind nonspecifically to the specimen of interest, they will be recognized by the labeled secondary antibody, resulting in higher background..... BSA and dry milk may contain bovine IgG. Secondary Ab directed against bovine, goat, horse, or sheep react with bovine IgG. Therefore, use of BSA or dry milk to block or dilute these antibodies may sinificantly increase background staining and reduce antibody titer. For blocking, normal serum (5%v/v) from the host species of the secondary antibody is recommended"
As for us, since we mostly use mouse or rabbit primary Ab with goat secondary antibody, the antibody diluting buffer always have BSA and FBS, sometimes (the good ones) will also have goat serum. We have never done blocking step, though, always after permeablization we go straight to primary Ab incubation. No problem for us.
We use FBS/NGS for blocking even if the secondary is rabbit or mouse or even goat or donkey.
Don't you get nonspecific binding when you use NGS for the secondary antibody like rabbit anti goat? I was thinking that the goat IgG in the goat serum binds to the sticky sites and results in a higher background.
Don't you get nonspecific binding when you use NGS for the secondary antibody like rabbit anti goat? I was thinking that the goat IgG in the goat serum binds to the sticky sites and results in a higher background.
Somehow we didnt have a problem. No explanations though.
i keep it specific
avidin/biotin block when i use avidin biotin
goat block when i use goat anti ... secondary
rabbit block when i use rabbit anti ... secondary
the same for donkey, horse etc
and i do wash between every step (only short washes to prevent mixing of chemicals)
dom
we use ngs/bsa in pbs for block and ngs in pbst for diluting abs. our abs are mouse monos, rabbit polys and goat antimouse or rabbit for secondaries.
we used to wash after blocking until we realized that it made no difference except to shorten the procedure if we didn't. so, now we don't.
I have always blocked in 5% BSA/PBS and will briefly rinse my slides in PBS before starting primary antibody. Doesn't matter though since I dilute my antibodies in the blocking BSA/PBS. I've only worked with mouse and rabbit antibodies previously but am starting to work with a third stain with a goat antibody. I didn't think about the potential for cross-reactivity to the BSA. Any other ideas for blocking? Milk doesn't seem to do much good for IF and I've tried fish skin gelatin but it's even worse than milk.
Actually I have used goat primary Ab before with the same FDB containing BSA and FBS and it still works for me. However, if you want to be careful, then use the serum of your secondary Ab to block.