Perfusion Fixative I

Making 200 mL of fixative for perfusion (1% GA, 4% PFA in 0.1M Phosphate Buffer)

• Heat 120 mL ddH2O to >60C<85C (do not boil)
• Add 8 g powdered Paraformaldehyde and immediately cover flask with foil to avoid toxic vapors (prepare in fume hood if possible)
• Clear with NaOH (one drop at a time, perhaps only one is necessary)
• Cool to RT with an ice bath
• Add 8mL of 25% Gluteraldehyde
• Add 50 mL 0.4M Phosphate Buffer
• QS to 200 mL with ddH2O
• Filter through a hydrophilic filter and aliquot into 50 mL tubes.
• Check pH and adjust to 7.2-7.4 with 1M HCl
• Store at -20C

Note: This fixative was designed to preserve muscle tissue. This protocol will also work with other buffers (PBS or Cacodylate) and with different percentages of fixative. Many times 2.5% GA is more appropriate.