Fluorophore – Page 3 – The Live Cell Imaging facility

Are there brighter versions of GFP or RFP out there?

Have you ever heard about Superfolded GFP? It is 50% brighter than GFP! And mScarlet is almost 6 times brighter than mRFP! How do I know? I look at this fantastic database called FPBase.

You can see which fluorescent protein is monomeric, sort them by excitation and emission or find which bleaches least or maturates fastest! Great tool! 🙂

Fluorophores are constantly being developed. If you make a new plasmid, make sure you check that the one your lab has been using for trillions of years is the very best one!

Skip the secondary, Part 2! :)

In one of the LCI earlier posts, you can read about easy and powerful it is to skip using a secondary antibody while still having a bright signal.

We have not had any feedback on the Kromnigon technology but we got to hear great praise and see superb images of a similar product called Mix-n-Stain by Biotium.

You can now directly label your stock of primary or favorite tag ligand (Snap, Clip, Halo, TMP) in just 30 min! Each primary gets 3-5 fluorophores according to the Mix-n-Stain brochure so there is no problem with dim directly labelled antibodies as used to be the case in older labeling technologies.

Skipping the secondary means gaining time but also no more headache about matching antibody and tissue species so you can stain your tissue with 7 or 8 antibodies if you image with narrow filters or spectral unmixing.

Bye bye ‘No primary’ controls! It is high time to switch to an isotype control: buy an antibody with the same isotype as your favorite antibody and label it in the same way. This allows you to detect any aspecific binding of your primary antibody.

Last but not least, skipping the secondary means less animals used to produce them. That alone is a bit plus!

It costs 100€ or so to label 50 ug of antibody. Definitely worth a try! 🙂

Skip the secondary! :)

Kromnigon makes fluorescent labels called FlexiStain that stick to biotin-labelled primary antibodies.

This means that you skip the species matching headache when using multiple antibodies and your labeling is faster! I have not tried myself but they offer free trial kits so give it a try! You are welcome to leave comments on this posts to tell us if it works or not! 🙂

Note: We have not had very good experience with these dyes. Check this post instead.

DAPI is so passé!! ;)

If you are tired of DAPI bleeding through your weak green channel but are stuck to using blue for the nucleus, you might want to give a try to Syto41.

Must better excitation efficiency at 405 nm and much narrower emission spectrum!

And Syto also comes in other colors. 🙂

Brilliant violet dyes

Those who work with FACS might be aware of the very bright dyes called Brilliant Violet. They can also be used in microscopy. 🙂

BV dyes are all excited around 400 nm (same as blue dyes) but they can emit at much longer wavelengths, like red or far red. This means that if they are used together, they are excited at the same time and the colors are only separated based on the emission! But these dyes are very bright which is a great advantage.

Et voilà! 🙂

Easy label of live cells

How to easily label Golgi, cytoskeleton, endosomes, lysosomes… in live cells with CFP, GFP or RFP then be able to fix everything? It does sound like pure magic! Have a look here.