The Intracellular Fixation & Permeabilization Buffer Set is designed for use in intracellular staining and flow cytometric analysis and has been specially formulated to reduce non-specific staining of fluorochrome-labelled antibodies and increase fluorescence signal to noise ratios. In the first step live cells can be “fixed” with the Fixation Buffer, which cross- links proteins. The second step, using the Permeabilization Buffer, creates holes in the membrane thereby allowing the intracellular staining antibodies to enter the cell effectively. Subsequent washing steps, antibody additions, and incubations after cell permeabilization should be performed using the Permeabilization Buffer. The final washing step and cell resuspension prior to running the sample is done using Flow Cytometry Staining Buffer (cat. 00-4222). This kit is commonly used for staining both cytoplasmic proteins as well as proteins in the secretory pathway such as chemokines and cytokines. It is not recommended for intranuclear staining. Instead, please refer to the Foxp3 Transcription Factor Buffer Set (cat. 00-5523) for nuclear staining.

Before the fixation step, it is recommended to label the live cells using one of the Fixable Viability Dyes (eFluor™ 450, 506, 660, 780, 520, or 455UV) and then proceed with fixation.

Reported Application
Flow Cytometric Analysis, Intracellular Staining Followed by Flow Cytometric Analysis