In the supplement it is described, that you use 2% horse serum for the incubation with the primary conjugated antibody. Also another question I have is whether you incubate with rotation or just let it stand at 4°C? I really hope, that you answer my question, it would be a great help.
Article Global Epigenomic Reconfiguration During Mammalian Brain Development
Hi
Incubation with normal serum is required to prevent non-specific binding of your primary antibody. The determination of the species is dependent on the species in which the primary and secondary antibodies were raised in.
For examples, if you are using a rat IgG primary antibody against a human protein, the secondary antibody against the primary must me from a third species (e.g. horse, goat, rabbit, etc) and to prevent non-specific binding of the primary IgG you use the serum of the third species to block all recognition sites except for your protein of interest.
Regards,
Bassem
Tahnk you for the fast answer!
In this case, they use anti mouse primary antibody that are directly conjugated to the fluorochrom. So actually just one species (they label mouse brain nuclei) and no secondary antibody.
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