"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 UniversityRating: 5.0
Examples A, B and C show some of the possible protocols used for double labeling two unconjugated primary antibodies from the same host species.
The success of these experimental designs will require some empirical manipulations. Optimizing reagent concentrations in each step or switching the labeling sequence of the two antigens may influence the outcome.
Step 1. After blocking with normal serum, incubate with the first primary antibody, in this example Rabbit Anti-Antigen X. Wash.
Step 2. Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibody, in this example unconjugated Fab fragment Goat Anti-Rabbit IgG (H+L). This presents the rabbit IgG as goat Fab. Wash.
Step 3. Incubate with conjugated tertiary antibody directed against the host species of the Fab antibody. The tertiary antibody must not recognize the host species of either the primary antibodies or the second secondary antibody. This example used Alexa Fluor® 488-Mouse Anti-Goat IgG (H+L) (min X Ms, Hu, Rb Sr Prot). Wash.
Step 4. Incubate with the second primary antibody, in this example Rabbit Anti-Antigen Y. Wash.
Step 5. Incubate with second conjugated secondary antibody, that does not recognize the host species of either the Fab antibody used in step 2 or the tertiary antibody used in step 3. In this example, Rhodamine Red™‑X-Mouse Anti-Rabbit IgG (H+L) (min X Hu, Gt, Ms, Shp Sr Prot) was used. Wash.
Step 1. After blocking with normal serum, incubate with the first primary antibody, in this example Rabbit Anti-Antigen X. Wash.
Step 2. Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibody, in this example unconjugated Fab fragment Goat Anti-Rabbit IgG (H+L). This presents the rabbit IgG as goat Fab. Wash.
Step 3. Incubate with conjugated tertiary antibody directed against the host species of the Fab antibody. The tertiary antibody must not recognize the host species of either the primary antibodies or the second secondary antibody. This example used Alexa Fluor® 488-Mouse Anti-Goat IgG (H+L) (min X Ms, Hu, Rb Sr Prot). Wash.
Step 4. Incubate with the second primary antibody, in this example Rabbit Anti-Antigen Y. Wash.
Step 5. Incubate with second conjugated secondary antibody, that does not recognize the host species of either the Fab antibody used in step 2 or the tertiary antibody used in step 3. In this example, Rhodamine Red™‑X-Mouse Anti-Rabbit IgG (H+L) (min X Hu, Gt, Ms, Shp Sr Prot) was used. Wash.
A detection method using conjugated Fab fragments and whole IgG secondary antibodies.
A blocking method to allow use of two same species primary antibodies following blocking for endogenous Ig with unconjugated fab fragments.
A blocking method using unconjugated fab fragments to prevent detection of the conjugated primary by subsequent antibodies.
A blocking method using whole Ig antibodies to prevent non-specific labeling.
Background staining may be observed if a labeled secondary antibody is not adsorbed to minimize recognition of endogenous tissue Ig.
View Blocking Protocol
FabuLight antibodies are Fab fragment secondary antibodies specific to the Fc region of IgG or IgM primary antibodies.
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