Molecular Probes® Tech Tips
Discover some of Molecular Probes' best-kept secrets with this frequently-updated list of fluorescence labeling and detection technical tips and hints from seasoned Molecular Probes® support staff.
Choosing organelle stains
If you're looking for a tool to help choose dyes for specific organelles, try the online Cell Staining Simulation Tool, where you can mix and match color and dye options for live and fixed cell labeling and see them in a simulated cell.
Labeling unpermeabilized cells
If you wish to label cell membranes in live or formaldehyde-fixed (but unpermeabilized) cells, be sure to specify plasma membrane or all membranes. Lipophilic cyanine dyes, such as DiI C18, will label all membranes of the cell, while wheat germ agglutinin conjugates, concanavalin A conjugates, or CellMask™ Plasma Membrane Stains are more selective for the plasma membrane.
Impact of serum on labeling
If you are labeling live cells with diacetate dyes, such as many ion indicators or cell tracking dyes, which are cleaved by cytoplasmic esterases to become active, make sure you do not label in the presence of serum, which has esterase activity and will prevent uptake of the dye.
Antigen retrieval techniques
Antigen retrieval techniques (used to unmask antigens in samples for immunolabeling) typically involve heating the sample but the best instrument, buffer, and timing differ from antigen to antigen. Here is one guide to different techniques: http://www.ihcworld.com/epitope-retrieval. Note: not all antigens require antigen retrieval.
Doing two-photon microscopy? Here is a good link for two-photon absorption cross-sections for dyes: http://www.drbio.cornell.edu/cross_sections.html
When choosing a dye, make sure it matches your filter specs or laser line. There are handy tools online. One is the Fluorescence SpectraViewer tool, where you can display the spectra for up to five dyes at a time and put in your filter or laser wavelengths to check for overlap.
- Also Available, the SpecraViewer iPhone™ and iPad™ App
Same species primary antibody tip
Have two primary antibodies of the same species for the same sample? To avoid cross-label, you can use isotype-specific secondaries if they are of different isotypes (IgG1, IgG2a, IgG2b), or you can make direct conjugates.
- Learn More About Antibody and Protein Labeling Kits
When choosing a primary antibody, be sure to check if it is validated for the technique you will use. It is typically noted in the product manual or certificate of analysis. For instance, antibodies validated for Western blotting or ELISA are not always successful for immunocytochemistry or immunohistochemistry techniques.
Checking secondary antibody concentration
When considering the range of concentration to test for your primary or secondary antibody, be aware that instrument sensitivity is also a concern. For instance, microscopy is typically less sensitive than flow cytometry and often requires a higher concentration.
Make sure to clean your objective very well if you change from one immersion oil to another. If two unlike oils mix, not only can there be refractive index problems, but some will react with each other to form a sticky substance that can damage your lens.
Is your fluorescent dye solution a slightly different color than older lots, when viewed by eye in room light? Don't worry, the color can vary in production from lot-to-lot, and isn't important for most fluorescent dyes. What matters is the fluorescent properties and functionality.
Secondary antibody background problems
Problems with apparent secondary antibody background? Be sure to run a secondary-only control to insure the problem isn't from the primary, try increased protein blocking (such as 6% BSA/10% normal serum), or reducing the secondary concentration or time, and consider that some background can be due to charge-based binding (which can be blocked with Image-iT® FX signal enhancer).
Insuring uniform field of view intensity
Having a uniform intensity across your field of view is important, particularly for quantitation. To correct for this, you can make a uniform intensity standard slide by making a solution of a reference dye in buffer and putting it under a coverslip, or purchase ready-made plastic uniformity slides from distributors.
Photobleaching problems? For fixed samples, using an antifade mounting medium is recommended (such as ProLong Gold®). For live samples, try using a second, more stable dye (such as Hoechst 33342) to find and orient the cell first, before switching over to your problematic dye to snap a picture, thus limiting the time it is being exposed and fading.
- Learn More About Antibodies from Invitrogen