Dear Lidia de Tapia

When one selects antibodies for flow cytometry, the first step one must consider is to check whether the antibody has been validated for the flow cytometry application. If this is not the case, the antibody cannot be assumed to work in this type of setting. Please note that each assay is different, targets are being examined under different conditions, and these conditions can affect the way the antibody will behave. When the application is meant for flow cytometry, it is only for flow cytometry. Though some antibodies having the flow cytometry application do work for confocal microscopy, but the results may not be up to the mark.

Another factor which you need to consider is the fluorophore conjugate. When you choose a fluorophore, you must take into consideration the available instrumentation and technique, like the flow cytometry, microscopy, or microplate reader. Some dyes are not suitable for some applications. For instance, APC, PerCP, and PE are typically used for flow cytometry and not microscopy because these dyes are not very photostable, and in imaging application photostability is important (e.g., rhodamine dyes usually display better photostability than most of the other fluorescent dyes).

So, I would not recommend flow cytometry antibodies for visualization in confocal microscopy.

Regards,

Malcolm Nobre

if the antibody clone has previously been shown in the literature to be well utilized for ICC/microscopy, the clone itself probably would work. The best evidence is if you can find papers from groups staining the same type of cells that you plan to stain. If there really is not a lot of evidence that the antibody clone has been used for ICC/microscopy, then its basically a gamble whether or not the antibody will work for ICC. Also, depending on how highly expressed or not the target antigen is, a directly conguated primary antibody could show dimmer results than an unconjugated ab which you would then stain with secondary abs. If you own the ab already give it a shot, if you don't own it and plan to use it only for microscopy, consider the unconjugated version and again do your due diligence to see if there is evidence that the clone 'works'.

In general, if antibodies work in tissue (confocal), they should always work in cell suspension (flow cytometry), but not vice versa.

Reasons are below:

1. Epitope availability. Some epitopes on the protein target might be blocked in tissue that cannot be accessed by antibodies using the same epitope. Then you may need to go through the "finding the right clone" process to make it work in tissue.

2. Fluorophore limitation. Many scopes are limited by the choices of laser they have, most don't have UV laser in 350nm, therefore you can't use BUV dye conjugated antibodies. BV dyes can be excited at 405nm but they are large polymer dye that may not be staining complex cell processes and tissue structures as well as small dyes such as Alexa Fluor dyes. Many flow antibodies are designed in these colors and not available in other dyes such as BD antibodies, while many tissue antibodies are limited in colors.

3. Non-specific signals. Regardless of the antibody clones and fluorophores, when using flow antibodies in tissue, there is always an unpredictable occurrence of non-specific signals that may prevent it from further analysis