"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 conjugated secondary antibody, in this example Alexa Fluor® 488-Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot). Wash.
Step 3. Incubate with normal serum from the same host species as the primary antibodies, in this example normal rabbit serum. The purpose of this step is to saturate open binding sites on the first secondary antibody with IgG so that they cannot capture the second primary antibody. Wash.
Step 4. Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibodies, in this example Fab Goat Anti-Rabbit IgG (H+L). The host species of the Fab antibody should be the same as the host species of the conjugated secondary antibody. This step covers the rabbit IgG so that the second secondary antibody will not bind to it. Wash.
Step 5. Incubate with the second primary antibody, in this example Rabbit Anti-Antigen Y. Wash.
Step 6. Incubate with the same secondary antibody as used in step 2, conjugated to a different probe, in this example Rhodamine Red™‑X-Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot). 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 conjugated secondary antibody, in this example Alexa Fluor® 488-Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot). Wash.
Step 3. Incubate with normal serum from the same host species as the primary antibodies, in this example normal rabbit serum. The purpose of this step is to saturate open binding sites on the first secondary antibody with IgG so that they cannot capture the second primary antibody. Wash.
Step 4. Incubate with an excess of unconjugated Fab antibody against the host species of the primary antibodies, in this example Fab Goat Anti-Rabbit IgG (H+L). The host species of the Fab antibody should be the same as the host species of the conjugated secondary antibody. This step covers the rabbit IgG so that the second secondary antibody will not bind to it. Wash.
Step 5. Incubate with the second primary antibody, in this example Rabbit Anti-Antigen Y. Wash.
Step 6. Incubate with the same secondary antibody as used in step 2, conjugated to a different probe, in this example Rhodamine Red™‑X-Goat Anti-Rabbit IgG (H+L) (min X Hu, Ms, Rat Sr Prot). Wash.
| Step 1. | - |
|---|---|
| Step 2. | - |
| Step 3. | - |
| Step 4. | - |
| Step 5. | - |
| Step 6. | - |
A detection method using conjugated Fab fragments and whole IgG secondary antibodies.
A blocking method using unconjugated fab fragments to “change” the species of one antibody.
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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