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Immunolabeling Hints

You can save a lot of time and money if you try to optimise your immunolabelling before coming to the microscopes. Here are a few hints to help?

  1. Work out the best labelling conditions for each primary antibody.

    • different fixation and permeabilization protocols
    • different concentrations of antibody
    • different incubation times and temperatures
    • different methods of blocking non-specific labelling

    These can make a big difference – for instance, microtubules are not preserved well by formaldehyde fixation, while phalloidin staining will not work on methanol-fixed cells.

  2. Try to pick the most appropriate secondary antibody conjugates for your experiment. This is particularly important for multiple labelling, for which you need to use conjugates that:
    • are as bright and photostable as possible
    • have well separated spectra (to avoid bleed-through)
    • have excitation/emission spectra that match the laser lines/filter sets of the microscopes
    • are optimized for multiple labelling by cross-absorption against sera from the other species
  3. We will be happy to discuss which fluorochromes work best for each of our microscope systems, and we have aliquots of a range of secondary antibodies and dyes that you can test out for free.
  4. If you need to counterstain your nuclei, you can image DAPI/Hoechst using all of our wide-field microscopes but only using one of our confocal microscopes (the others have no UV laser). For confocal work we recommend using the far-red DNA stains, Draq 5 or TO-PRO 3, instead. We can supply samples of these for testing.
  5. Use glass coverslips that are no. 11/2 in thickness. This is very important and yet many people still use no. 1 coverslips. Most modern microscope objective lenses are optimised for glass that is 0.17 mm thick, and no. 11/2 coverglasses correspond most closely to this figure. If you use thinner or thicker coverslips you can get spherical aberrations and greatly reduced fluorescence.
  6. For any high resolution work or for experiments where 3-D reconstruction is required, try to avoid using slides with wells. The further your sample is from the coverslip, the worse the spherical aberration can be. In addition, you may be unable to use many of our high numerical aperture objectives as these typically have a short working distance.
  7. Think carefully about the best mounting solution for your sample. Decide in advance whether you require a semi-permanent mount, which may compromise your staining intensity and any 3-D reconstruction of your images, or whether you will be collecting your images within a few days. If your staining is weak and prone to photobleaching, include an anti-fade reagent. However, be aware that particular fluorochromes are more compatible with certain anti-fade reagents than with others. The Molecular Probes catalog is a good source of information on this topic.And finally…If any of you have spectacularly good or bad results using a particular reagent, please let us know so that we can disseminate this information to other members of the University. We will post new recommendations and suggestions on our Announcements page – so keep a look-out!


The Rockefeller University
1230 York Avenue, Box 209
New York, NY 10065

Bio-Imaging Resource Center
Bronk Laboratory
DWB Room 201 (office)
DWB Room 202, 203, 102, RRB 154A (Lab)