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The choice of primary antibody is critical to the success of the IHC experiment. The most important feature of the primary antibody is its specificity for the target antigen. The antibody is then detected either directly, through a label that is directly conjugated to the primary antibody, or indirectly, using a labeled secondary antibody that has been raised against the host species and antibody type and subtype of the primary antibody. The antibody is visualized using either a fluorescent label or an enzyme that converts a soluble substrate into an insoluble chromogenic product.
The steps in a typical immunostaining protocol are shown below. In direct immunostaining methods, the incubation step with the secondary antibody is omitted. The last step, where substrate is added, is omitted in fluorescent detection.