Frequently Asked Questions (FAQ)
/x-tad-bigger>Associate Professor of Clinical Pathology
Institute for Cancer Genetics, Columbia University
New York, USA
Do I need detergent to permeabilize the cells?
Once cells are fixed (in suspension, smeared on a slide, cytospun, inside a piece of tissue) they no longer can select intracellular traffic of molecules . In addition, processes such as drying and lipid solvent treatment (acetone, ethanol etc.) creates huge holes in the subcellular structure.
Antibodies can easily cross cell membranes (cytoplasmic, nuclear) in fixed cells.
Whether they can reach the
antigen in case of fixation dependent antigen masking, is another
problem. No detergent is able to do antigen unmasking.
The crucial role of low levels
of detergents such as Tween 20 is to reduce surface tension in
washing solutions, allowing the slides to remain wet.
There is no role for detergent in permeabilization of fixed cells. Only exceptions are:
saponin treatment of living cells for flow cytometry staining
high concentration detergent mediated extraction of nuclear / cellular matrix-associated substances in lightly fixed cells.