- What is the excitation and emission wavelength of the fluorochrome?
- Which product would be a suitable isotype control for this antibody?
- Do the samples need to be fixed? Do I fix before or after staining?
- Do the cells need to be permeabilized?
A list of fluorochromes with their excitation and emission wavelengths can be found here:
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 isotype control on our website, use the product browser on the left hand side of the home page. Choose "isotype control", then the species and subclass of your primary antibody. This will give you a range of isotype controls for use with that species and isotype of primary antibody.
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. However, we do provide several polyclonal isotype controls for use in these circumstances.
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 4oC 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 guide.
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 guide