Re: Ideas for good, unusual labels for fixed cells? *vendor reply*

[Kilgore, Jason A.]

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** vendor reply **

If the cells are fixed, but not permeabilized yet, you can label with a WGA conjugate for the plasma membrane, then permeabilize and label with other dyes/antibodies. There are also various different colored nuclear dye options in different wavelengths. The most selective in each wavelength for fixed cells include: DAPI or Hoechst 33342 for blue, SYTOX Green for green, 7-AAD for red (but it is wide in spectrum, so maybe not), or TO-PRO-3 or DRAQ5 for far red.

But if you label live, and THEN fix the cells, it opens up a lot of options for you, including:

- CellLight reagents, as long as your cells are not hematopoietic in nature, can transduce your cell over night for a number of different organelles and structures and is retained after fixation and permeabilization. GFP, RFP, and CFP options are available for many targets. They all share the same product manual, which has a comprehensive table of organelle and color options to choose from.

- MitoTracker Red CMXRos, MitoTracker Orange, or MitoTracker Deep Red, for mitochondria (MitoTracker Green FM is not fixable)

- WGA conjugates for plasma membrane. Concanavalin A conjugates also work well.

- LysoTracker Red (lysosomes) (other LysoTrackers are not retained with fixation)

- ER-Tracker Blue-white (ER-Tracker Green and ER-Tracker Red are also fixable but, unlike the blue-white version, don't survive permeabilization well).

- the various labeled 3K or 10K MW dextrans, as long as they are labeled "fixable" or "lysine fixable", which can be taken up into endosomes and fixed in place afterward.

Please let me know if you need further help,

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Life Sciences Solutions
 

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Subject: Ideas for good, unusual labels for fixed cells?

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Dear listers,

we are about to set up a multi-color experiment as proof of principle in formaldehyde fixed mammalian cultured cells. Probably HeLa or CHO.

We are planning on DNA stain, phalloidin as actin stain, some GFP label (amplified with nanobodies), and two primary antibodies from rabbit and mouse, e.g. for mitochondria , ER, nuclear pores or the like with the fluorochrome on the secondary ab. The actual labeled structures do not matter so much, as long as they give good signals of specific structures (i.e. not just a cytoplasmic, diffuse protein.) Our secondaries are from goat (or donkey)

We would like to stain one or two additional structures, but we are not sure how to label them. Antibodies from rat have a tendency to cross-react with mouse antibodies, that would not be ideal. (to be
precise: the secondary antibody cross reacts to the other species, of
course.)

Does anybody have some ideas for additional good labels? An antibody from chicken (or other species) that nicely delineates the Golgi, ER, Mitochondria, focal adhesion points, nuclear pores, splicing factors, etc, would be great.

Any good antibody with a cellular target where the secondary will not cross react with mouse or rabbit ab would be of interest. Or some good non-antibody labeling of cellular structures, like phalloidin, just for something other than actin or DNA, that gives decent signals in fixed cells.

Of course on could go for labeling of various primaries from the same species. But antibody labeling is not exactly our core competence and it would add one more layer where things can go wrong. Plus, we might not get the fluorochromes that we plan to use.

Hoping on your experience

Steffen

--
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Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
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