Hello all, I have a primary antibody produced in rat, that I would like to test on rat cryosections. However I am afraid that the secondary antibody (which will be anti-rat) produces a high background because it is anti-rat on rat sections. Does anyone have protocol to avoid this problem of species reactivity ? except from buying another antibody!! Maybe enhancing the blocking steps and/or washings, to avoid non-specific fixation?
Many thanks
You can try blocking overnight, but you really should use an antibody that is raised in a host that isn't the same as your tissue species. It is the only way to make sure that you don't have cross-reactivity with endogenous immunoglobulins in the tissue.
Although I never tried them:
There are mouse-on-mouse staining kits available - maybe you are lucky and it also works with rat-on-rat?
Hi Anne,
In my opinion, the best blocking strategy would be to use an anti-rat Fab fragment prior to your primary antibody incubation. You can buy these from Jackson ImmunoResearch. See here for example: https://www.jacksonimmuno.com/catalog/products/112-007-003
Since these unlabelled anti-rat Fab fragments have only one binding site, they should get attached to the endogenous rat antibodies, without interfering (i.e. capturing) your rat primary antibody. If you fear that these fragments get detached during the washes, you can do a quick (10min) incubation in 4% formaldehyde after this blocking step to fix the fragments in place. The dilution at which you should incubate these Fab fragments needs to be empirically optimized, but I would say from 1:10 to 1:100.
An other possible problem is that the Fc receptors that are present at the membrane of some cells would capture your rat antibodies (although they are likely to be ineffective due to formaldehyde fixation). To avoid this, an incubation with rat serum might be required, before the blocking with the anti-rat Fab fragment.
In summary, I would suggest that you first try your anti-rat secondary antibody alone, to evaluate the amount of unspecific staining. Then, try some of the blocking steps I suggested above, the best being (in my opinion): 1) Incubation in rat serum to block the Fc receptors. 2) Incubation in an excess of unlabelled anti-rat Fab fragments to block the endogenous and "serum-added" rat antibodies. 3) 10min fixation in 4% formaldehyde, to avoid all this stuff from moving around anymore. 4) Proceed with your primary and secondary antibody staining.
It's up to you to decide whether this is worth the trouble. I agree with Leah Schneider that you really should try to find an antibody from an other species, it would be the safest (and porbably quickest) way to go.
Good luck anyway!
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