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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
It is most effective to block tissue or cells with normal serum (5% v/v) from the same host species as the labeled secondary or tertiary antibody. The IgG in serum should occupy sticky sites on the tissue or cells to prevent non-specific binding of the labeled IgG antibody.
Never block the tissue or cells with normal serum from the same host species as the primary antibody. For example, if a primary antibody is made in mouse and normal mouse serum were used for blocking, the mouse IgG would bind to the sticky sites and be recognized by labeled anti-mouse IgG. A higher background would result.
Two commonly used blocking reagents are bovine serum albumin (BSA) and dry milk. However, most commercially available BSA and dry milk products are contaminated with bovine IgG, which can cause a problem when goat or sheep primary antibodies are used. A labeled anti-goat secondary antibody will significantly cross-react with bovine IgG since goat, sheep and cow are closely related species.
These principles apply to all immunological procedures including ELISA, Western blots, flow cytometry, immunohistochemistry, and immunocytochemistry.
Choose a labeled secondary antibody cross-adsorbed against the species of the experimental tissue, if possible. For example, if the tissue is human tonsil and the primary antibody is made in mouse, use a labeled anti-mouse IgG that has been adsorbed against human, such as Alexa Fluor® 488-Goat Anti-Mouse IgG (H+L) (min X Hu, Bov, Hrs Sr Prot) (115-545-062).
If mouse tissue containing immunoglobulins is probed with a primary antibody made in mouse, the endogenous immunoglobulins should first be blocked with a monovalent Fab fragment of anti-mouse IgG.
Avoid overloading your washing chamber with too many slides.
Inadequate washing may also be a problem when labeling a whole mount or a thick section that requires a longer incubation time. The time required for unbound antibodies to diffuse out should be comparable to the time needed for antibodies to penetrate into the sample. For instance, use 3 x 20 minute washes following a 60 minute incubation. The washing step may need to be further extended depending on the specific sample.
For example, if a labeled anti-goat IgG is diluted in buffer containing IgG-contaminated BSA or dry milk, sticky immune complexes can form due to cross-reaction of anti-goat IgG with bovine IgG. Diluting the antibody in wash buffer only, for example PBST, is preferable to diluting in blocking reagent. For more effective blocking, incubate with blocking reagent as a separate step instead of adding it to the antibody diluent.
Titrate the labeled secondary antibody to obtain a maximal signal to noise ratio. Labeled secondary antibodies can be diluted further than expected.