Immunohistochemistry and Immunocytochemistry Frequently Asked Questions

• Immunohistochemistry
• eFluor® Nanocrystal Conjugation Kits

Immunohistochemistry

Q. I am interested in staining tissue sections. Are your antibodies suitable for this application?

We are in the process of testing our antibodies in house on IHC applications. Please contact eBioscience Tech Support for additional information that may be available.

Q. Do you have protocols available for your antibodies and immunohistochemical staining?

We do have general IHC protocols available for frozen tissues and paraffin embedded tissues. For clone specific information, please contact eBioscience Tech Support.

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eFluor® Nanocrystal Conjugation Kits

Q. When performing immunofluorescent staining on cells and tissues, are there any changes to the protocol?

Please refer to our recommended protocols in the Best Protocols® section of our website.

Q. My staining works fine with purified followed by a secondary antibody, but the eFluor® Nanocrystal conjugated antibody does not appear to be working. What could be wrong?

Although the protocol works for two-step or three-step staining, the conditions may need to be changed for staining with eFluor Nanocrystal conjugates. Here are several points to consider:

1. Protocol - We recommend using the protocols developed by eBioscience that have been shown to work for a variety of direct eFluor Nanocrystal conjugated antibodies. Please refer to our recommended protocols in the Best Protocols section of our website.
2. Blocking buffer - We have formulated two versions: one containing high amounts of protein and another with lower levels. We recommend starting with IHC/ICC Blocking buffer-Low Protein (Cat. No. 00-4953), which has been shown to be optimal for general use when staining cytoskeletal, cytoplasmic and membrane proteins. For improved specificity and better signal-to-noise ratio, the IHC/ICC Blocking Buffer - High Protein (Cat. No. 00-4952) has been found to give superior results especially with antibodies to nuclear antigens and FFPE tissue.
3. Incubations times - For best results, we recommend blocking non-specific sites for 1 hour at room temperature and incubating eFluor Nanocrystal conjugates overnight in blocking buffer at 2-8°C protected from light.
4. Mounting media - Please refer to table below on selection of mounting media by application.

Q. Will eFluor® Nanocrystal conjugated antibodies blink?

Although nanocrystals can blink, all efforts are taken during manufacturing to prevent this.

Q. When performing immunofluorescent staining on cells and tissues, are there any changes to the protocol?

Please refer to our recommended protocols in the Best Protocols section of our website.

Q. What should my starting concentrations be?

All antibodies should be titrated for optimal performance. The final antibody conjugate concentration from both the Amine and Sulfhydryl-Reactive kits should be in the range of 3-6 uM for the eFluor Nanocrystal 605 and 650 conjugates. For ICC and IHC applications, we recommend a starting concentration of 1-40 nM, although the optimal concentration must be determined for the cells or tissue of interest.

Q. How should I mount my slides?

Special Note: For experiments requiring extended illumination times or using organic dyes in combination with eFluor Nanocrystal conjugates, it may be necessary to mount stained tissue or cells with mounting medium containing an anti-fade component. We suggest using Vectashield mounting medium for these conditions.

Q. How should I store my slides?

Slides should be sealed with clear nail polish and stored at 2-8°C in the dark.

Q. How long do I have to look at my slides after completing the staining?

A benefit to using the nanocrystals is that they do not photobleach like organic dyes. If mounted and stored properly, the slides will be good for weeks, if not longer.

Q. Which buffers and detergents are compatible with the eFluor Nanocrystal conjugated antibodies?

Because you are working with directly conjugated antibodies, there should not be a need or detergents in your buffers. It is best to avoid their use if possible. We recommend using Tris Buffered Saline (TBS), pH 7.4 (Cat. No. 00-4954) rather than Phosphate Buffered Saline (PBS) for washing following binding of eFluor Nanocrystal conjugated antibodies.

Q. Can I use a PAP pen?

No, we find that traditional PAP pens are not compatible with eFluor Nanocrystal conjugated antibodies.

Q. What filter sets do I need to detect the eFluor Nanocrystals for microscopy?

For best results use the filter sets listed above. Additional filter sets may be usable for the fluorophore of interest, but are likely to result in decreased signal intensity.

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