Abridged from Jacoberger JW, 1991, Intracellular antigen staining: quantitative immunofluorescence. Methods 2:207-218.

Fixation Method Reagents Use Advantages Problems
Organic Solvent Fixation EtOH
MeOH
Acetone		 Long time storage

Inactivate cellular processes		 No covalent modification of DNA, RNA, protein, or carbohydrate epitopes

Permeabilizes cells		 Not good for lipophilic antigens

Cell aggregation
Formaldehyde Fixation Paraformaldehyde Fixation for surface or intracellular staining when followed by lipid solvents Relatively mild fixative

Cell membrane integrity preserved		 Severely broadens DNA histogram peaks when using PI simultaneously with cell surface or intracellular staining
Detergent Lysis Triton X-100
Tween 80
NP 40
Saponin		 Rapid permeabilization

Native conformation of intracellular antigens

Requires fewer washes, preserving limited cell numbers		 Rapid

Requires fewer washes		 Cell fragility

Loss of cytoplasmic cell components

High debris component

Limited storage time
Freeze/Thaw Freeze/Thaw buffer Same as detergent lysis Same as detergent lysis Same as detergent lysis
Formaldehyde/Detergent Paraformaldehyde
Triton X-100		 Primarily used for simultaneous nuclear antigen and DNA content

Detection of antigens in all cell compartments at higher formaldehyde concentrations		 Good DNA histogram CV at low formaldehyde concentrations

Low debris component

Few aggregates		 Loss of loosely bound cellular components

Short-term storage
Formaldehyde/Methanol Paraformaldehyde
MeOH		 Retains antigen within the cell better than alcohol alone Cells can be stored indefinitely

Antigens retained better within cell

Low debris component

Few aggregates		 DNA histogram CV higher than alcohol alone