Roy J. Carver
Biotechnology Center

Formaldehyde creates bonds between lysine residues to cross link proteins

Alcohol fixative - usually 70% ethanol or methanol

• PHA Concentration: 0.5% to 4% in PBS
• Stable for 1-2 weeks at 4°

• Dissolves lipids (no need for permeabilization)
• Can be stored at -20° for months.

Note: Do not use to measure GFP (fix with PHA then permeabilize with ethanol).

Permeabilization agents can be detergents or alcohols

• Detergents (choose wisely as if done insufficiently there will be no staining;  high levels of background if unbound antibody cannot be washed out sufficiently).
• Weak detergents:
  • Digitonin
  • Saponin
• Harsh detergent (used for nuclear membrane):
  • Triton X-100
• Large fluorophores will need more washes (PE and APC).
• Small proteins can leak out.