Improve your signal by using more antibodies, not more antibody.
If you have a marker that doesn't stain well, sometimes you can improve the detection by using two different antibodies against the same marker. This works if the antibodies recognize different epitopes on the same protein, allowing you to stack more signal per protein.
This isn't a new idea, by the way: people have used this approach for detection of CD25 in human samples to get around variability and poor staining. In a published example, Presicce et al. looked at differences in anti-human FoxP3 antibody clones and how stacking increased detection of Tregs:
Here's the type of improvement you can get, using mouse Foxp3 as an example:
Note that the %Foxp3+ is slightly lower for the REA788, largely due to some of the lowest expressing cells falling out of the bottom of the gate.
Here's a summary of the median intensity of Foxp3 expression in the Tregs:
I primarily use this approach for low expression transcription factors. In addition to Foxp3 (mouse and human), Bcl-6 and Nur77 work quite nicely with this approach. Bcl-6 stacking is especially helpful for identifying Tfh, which express lower levels of Bcl-6 than germinal center B cells.
Staining for Bcl-6 using Bcl-6 AF647 on mouse CD4 T cells:
Foxp3 clones: FJK-16s and REA788.
Bcl-6 clones: K112-91 and IG191E/A8. REA373 can also be used as a third clone.
Nur77 clones: 12.14 and REA704.
How can we know which clones are stackable?
In order to get additive signal from using two or more clones, they must recognize different epitopes on the same protein. This needs to be determined empirically, but fortunately, the scientists at Miltenyi have done this for their recombinant REA antibodies. These comparisons between the REA and other commonly used clones show the degree of overlap in specificity, and from this we can infer the epitope specificity between the non-REA clones as well. Here's an example for human FoxP3 from Miltenyi's website (scroll down to Extended Validation).
How do we determine how much of each antibody to use in a stacking combination?
Titration. First, titrate each antibody separately to determine the optimal concentration. Next, perform a matrix titration with the two (or three) antibodies at concentrations near the optimal point. For example, if the optimum for clone A is 1:500 and clone B is 1:100, test combinations of A at 1:500, 1:1000 and 1:2000 with B at 1:100, 1:200 and 1:200 (3x9 = 9 total combinations). Usually, the best results in combination will be obtained with optimal or half-optimal concentrations.
Comments