Your colleagues submitted great questions during the webinar, and we’ve collected and answered them here in the following Q&A:

Controls

For the direct conjugate strategy, what would be the proper negative control?

There isn't a good, on-hand negative control. A labeled nonspecific IgG of the host species would probably be best.
During IF staining, is it necessary to have a negative control that has no primary antibody but everything else?
It is always a good idea to have controls. No biological protocol is infinitely repeatable. You must have contriols and optimize for every staining experiment. Without controls, you have no troubleshooting tools.

Multiplexing

In double- or triple-labeling experiments, is there any problem with incubating all of the primaries in the same step?

The answer is maybe. If you have used all of the antibodies together before with no cross-reactivity, you can make a "cocktail," but you need to assure yourself there is no other cross-reactivity in pilot studies.

Is it possible to combine labeled primary antibodies with other same-species antibodies which would be secondary-labeled with different dyes in a multiplex protocol?

No, there is no way for the anti-mouse secondary to target the "correct" mouse antibody.
For multiple labeling, can you put two or more primary antibodies together?
It depends on whether the antibodies are from the same species or not. For example, you cannot combine multiple rabbit antibodies, and then expect an anti-rabbit to find the "correct" one in the mix.

Blocking

What kind of blocking reagent is good for fluorescent labeling?

Our most common block is 4% IgG-free, Fraction IV BSA in PBS. But you can use other proteins, or even serum from the host species.

Is there a special blocking agent used to block endogenous biotin?

Life Technologies offers the Endogenous Biotin Blocking Kit. Essentially you use an unlabeled avidin to block endogenous biotin, then flood that with free biotin to cover all the binding sites on that avidin. Then carry on with antibodies.

Is there a special blocking agent used to block endogenous peroxidase when using TSA amplification?

That can usually be accomplished with extra H2O2 incubation, or a commercial prep blocking solution.

Do you suggest using BSA or casein as a blocking solution?

BSA is much more common in cell imaging. Casein is mostly a western blot block.

Sensitivity

How many dyes per avidin?

There are 3 to 4 dyes per avidin, and an average of two avidins binding to a given biotin antibody.

For secondary labeling, can sensitivity be increased by using higher concentrations of secondary antibody?

Up to a point, yes. However, you can only bind so much secondary antibody to your primary antibody. After that, you get dye-sample interaction and just background signal.

Secondary Compatibility

Does my secondary have to be from a species different from that of my tissue?

That's usually a good idea. There is a risk of Fc receptor recognition if you use a same-species antibody.

if you need to use chicken anti-goat and goat anti-rabbit secondaries, is it possible to prevent them from binding to one another?

Not easily. Your best approach would be to use the goat primary; then the anti-goat secondary; then block and use the other antibodies. You must reduce the exposure of the anti-goat to the other goat antibody.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.