2% GA in 0.05M PB, 0.1M Sucrose

2.5% GA, 4% PFA in 0.1M CB, 0.1M Sucrose

5% Gluteraldehyde in 0.1M Cacodylate Buffer

1% OsO4 in buffer

2% OsO4 in buffer

2% GA, 2% PFA in 0.1M Cacodylate Buffer

2% GA, 2% PFA in 0.1M Phosphate Buffer

Perfustion Fixative II

Making 200 mL of fixative for perfusion (2.5% 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 20mL 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

3% Paraformaldehyde for IEM

Mouse Glutaraldehyde Fixative (muscle)

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.