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Flow Cytometry (FCM) /FACS | FAQ

1. What fluorochromes can I use? 3. Do the cells need to be permeabilized?
2. What controls do I need? 4. Do the samples need to be fixed?

1. What fluorochromes can I use?

There is a guide for "Fluorochrome Selection".

2. What controls do I need?

Isotype controls are used to confirm that the primary antibody binding is specific and not a result of non-specific Fc receptor binding or other protein interactions. The isotype control antibody should match the primary antibody's host species, isotype, and conjugation. For example, if the primary antibody is a FITC-conjugated mouse IgG1, then you will need to choose a FITC-conjugated mouse IgG1 isotype control.

To find a suitable experimental control on our website, please see "Recommendations for Background Control".

Isotype controls for polyclonals
Most isotype controls are monoclonal. They are not suitable for use with polyclonal antibodies as these contain more than one IgG subclass.

3. Do the cells need to be permeabilized?

We recommend checking the datasheet for further details. If the target protein you are detecting is a membrane protein, then it is most likely that a permeabilization step will not be required. On very rare occasions, the antibody epitope may be in the cytoplasmic domain of the membrane protein. In this case, you may need to permeabilize with a gentle permeabilization agent, such as saponin. Check the data available on the datasheet (Abreviews and other images) to see if other researchers have required a permeabilization step. All cytoplasmic and nuclear target protein will require cell permeabilization for detection by the antibody.

You can view our "intracellular staining protocol".

4. Do the samples need to be fixed?

Any samples which contain potentially biohazardous material should be fixed. We would recommend adding 1 to 2% paraformaldehyde to the sample once staining is completed. The samples should then be kept at 4℃ in the dark and analyzed within 24 hours.
For intracellular staining, the cells are fixed and permeabilized together through the procedure. See our "intracellular staining protocol".

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