Is agitation necessary during IHC antibody incubation?

Agitation is not necessary during antibody incubation in IHC, since antibody dilution buffers will generally contain Tween-20 or Triton X-100, which are both detergents that help permeabilize the cell membrane and help with penetration of the antibodies. Agitation also has the potential to damage tissue, which is why it should be avoided.