Cell Fixation and Permeabilization Protocol using 70% Ethanol


Protocol Steps

Cell Fixation and Permeabilization Protocol Using 70% Ethanol


  1. Prepare 70% Ethanol and chill to -20°C.
    Tip: Do not freeze ethanol for long-term storage.  

  2. Prepare target cells of interest and wash 2X with PBS, centrifuging at 350xg for 5 minutes

  3. Discard supernatant and loosen the cell pellet by vortexing.  

  4. Add 3ml cold 70% ethanol drop by drop to the cell pellet while vortexing.  

  5. Continue vortexing for 30 seconds and then incubate at -20°C for 1 hour.

  6. Wash 2X with BioLegend Cell Staining Buffer (Cat. 420201) and resuspend cells at 0.5-1.0 x 107 cells/ml.  

  7. Use 100µl cell suspension/staining tube.
    Note: Please note that certain markers or fluors may not survive ethanol fixation. Protein-based fluors, like PE and APC, tend to have more difficulty, while synthetic fluors, like Brilliant Violet™, tend to have a higher chance of surviving the process.  



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