Are antibodies labelled with fluorescence that have not attached to an antigen visible under light microsocopes?

Question

I came across this thought while studying histology, what happens to fluorescence labelled antibodies that do not bind with an antigen, can we see them? or are antibodies activated upon antigen binding which in turn activates the Fc region to which the fluorescence molecule is attached.

Answer 1

By far the most common IHC protocol, in my experience, is using a primary antibody which binds to your target of interest, followed by a secondary antibody (which carries the fluorescent tag or labeling enzyme) to bind the primary antibody.

An example protocol from one manufacturer is found here:

https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-paraffin-embedded-tissue

Summarizing:

4. Apply primary antibodies diluted in Incubation Buffer according to manufacturer’s instructions...

5. Wash slides 3 times for 15 minutes each in wash buffer.

6. Incubate with the NorthernLights™ secondary antibody diluted in Incubation Buffer according to the manufacturer’s instructions...

7. Wash slides 3 times for 15 minutes each in wash buffer.

The "wash" steps are intended to remove from the tissue any primary antibodies that are not bound and later any secondary antibodies that are not bound.

You may still get some residual background fluorescence either due to non-specific binding of the secondary antibody, or unbound primary antibody that isn't washed out (I believe in typical use the non-specific binding of the secondary antibody is going to be the most relevant contributor to background, but I don't know a good citation for that), but you use "blocking" steps to attempt to reduce non-specific binding, and the assumption is that anything remaining contributes to a fairly even background stain.

You can further use negative controls that omit the primary antibody to estimate non-specific binding of the secondary antibody and check for any unexpected partitioning that may contribute to an artifactual signal.