Antibody Fixation and Intracellular Staining Considerations

eBioscience Clone Performance Following Fixation / Permeabilization

It is possible that fixing cells prior to staining can alter the antibody-binding epitope on a protein. This alteration of the epitope may affect the antibody’s binding affinity. Therefore, we have investigated the effects of fixation and permeabilization using our Intracellular Fixation & Permeabilization Buffer Set (cat. no. 88-8824). The activity of eBioscience antibodies following fixation is summarized in the table below.

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Intracellular Staining Buffer Selection

When performing intracellular staining for flow cytometry the selection of buffers used for fixation & permeabilization has a significant impact on the quality and accuracy of data. eBioscience provides optimized solutions for staining cytsolic, nuclear and secreted proteins for flow cytometry. The Foxp3 Staining Buffer Set is optimized for staining all nuclear factors tested to date, including all transcription factors, whereas our conventional intracellular staining buffer system, eBioscience Fixation & Permeabilization is recommended for staining cytosolic and secreted proteins for flow cytometry. We have investigated the compatibility of the Foxp3 Staining Buffer Set (cat. no. 00-5523) versus the Intracellular Fixation & Permeabilization Buffer Set (cat. no. 88-8824) for situations where simultaneous staining of both nuclear factors and cytosolic/secreted proteins is required.