Controls for Imaging Multiple Fluorophores

CITE requires all users to bring the appropriate control samples to your training session, so we can show you how to collect these images and advise you on any issues that arise with the controls.  If you are unable to prepare these samples in time for your training, please let us know before you arrive for training.

For your proposed experiments, please bring the following controls to your training in addition to your experimental samples:

1. Autofluorescence control: A sample that has been fixed, extracted and blocked in the same manner as your experimental sample, but without any fluorphore added.
2. Secondary antibody non-specific binding control: For experiments using fluorescently-labeled secondary antibodies, a sample that has been fixed, extracted and blocked in the same manner as your experimental sample, but treated with secondary antibody only (ie, skip the primary antibody step).
3. Bleed-through controls: Bleed-through is when the signal from one of the fluorophores is visible in the channel for another fluorophore. For example, you are looking at your sample with the Alexa 488 filter cube, but the small amount of green signal that Alexa 568 generates is visible as well. Whether or not bleed-though is visible depends on the sample, the microscope, the fluorophore and the quantity/intensiy of the labeled component – you therefore must test for bleed through for each experiment in which one of these variables changes. For each of the fluorophores you plan to use, bring a sample that has been labeled only with one primary and secondary pair.  For example, if you are imaging a sample labeled with antibodies labeled with Alexa 488 and Alexa 568, a “488 only control” would be labeled with only the primary antibody and Alexa 488 secondary.

Here is a summary chart of the controls you need for an immunofluorescence experiment with 3 labeled components: Primary antibody # 1 and fluorophore #1; primary #2 and fluorophore #2, and primary #3 and fluorophore #3.  For ALL controls, you should fix, extract and block the samples.