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. 🙂
Remember that with passive clarity, you can make most tissues transparent in order to image them.
The challenge may be to get the antibody to penetrate fully if your tissue is very thick but clarity removes lipid so it also helps with antibody penetration. If your protein expresses a fluorescent protein, you skip this problem and clarity preserves fluorescence.
You can then mount your tissue and image it on our microscopes! 🙂 It’s magic!! 🙂
There are loads of multiwell plates and chamber slides on the market but it is hard to get any that fulfill these criteria:
- They do not kill the cells (unfortunately, this is not a joke!)
- They do not leak (no joke either! Sigh…)
- They have a low skirt: so they can be completely imaged (including outer part of outer wells) with our Nikon 20x air, NA 0.75, WD 1mm objectives. The ‘skirt’ is the rim at the edge of the plate. A high skirt raises the plate so that it becomes impossible to image the wells at the edges of the plate as the objective bumps into the insert edge.
- They have a #1.5 glass bottom
- They don’t cost a bomb.
We recently found a great source of plates and chambers that fulfill all these criteria and are very flat! Check the Zell Kontakt website (email under Legal disclaimer)! 🙂
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! 🙂
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.