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"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."
Janet Duerr, Ohio University
Background staining may be observed if a labeled secondary antibody is not adsorbed to minimize recognition of endogenous tissue Ig. When a primary antibody is the same species as the tissue under study (e.g. mouse primary used on mouse tissue), blocking endogenous Ig suppresses the off-target signal.
To block endogenous immunoglobulins on cells or tissue sections, incubate with an excess (20-40 µg/ml) of unconjugated Fab anti-IgG (H+L) antibody just after blocking with 5% normal serum.
Blocking efficiency can be confirmed by eliminating the primary antibody from the protocol and incubating with
It may be necessary to increase the concentration of Fab antibody up to 100 µg/ml to suppress signal from high levels of endogenous IgG. To avoid displacement of the Fab antibody by the labeled secondary antibody, a light cross-linking with glutaraldehyde may be necessary, provided that it does not affect antigenicity of the target proteins.
N.B. Fab antibodies are not effective for blocking immunoglobulins in Western Blotting or ELISA applications.