Fixation
- Cut out the mouse liver and place it in a small amount of buffered glutaraldehyde on a piece of dental wax. With a clean razor blade cut the liver into small pieces, about 1mm2. The smaller the pieces the better the penetration of the fixative throughout the tissue.
- Drop into fixative: 3% Glutaraldehyde in 0.1M Phosphate buffer, pH 7.2. Fix for 1 hour at room temperature.
- Rinse sample in buffer at least 3 times for 15 minutes each. An overnight rinse in buffer before post-fixation in OsO4 is also acceptable.
- Post-fix the sample with 2% OsO4 in 0.1M Phosphate buffer, pH 7.2. Post-fix for 1 hour at room temperature.
- Dehydrate sample through a graded ethanol series.
50% EtOH 15 min.
70% EtOH 15 min.
95% EtOH 15 min.
100% EtOH 20 min. x 3 changes
Propylene Oxide 15 min. X 2 changes
1:1 P.O./Epon mix Overnight - Embedding of sample material in pure Epon (Complete mix). See Luft, 1961.
INGREDIENT Small Batch Regular Batch Large Batch
Epon 812 4.4 gms. 8.8 gms. 17.6 gms.
DDSA 3.1 gms. 6.2 gms. 12.4 gms.
NMA 1.8 gms. 3.6 gms. 7.2 gms.
DMP-30 0.13 gms. 0.26 gms. 0.52 gms.
Mix thoroughly with an applicator stick. Don't create air bubbles. Be gentle with stirring. Use disposable vessel that will not react with Epon ingredients. We use polypropylene beakers (S/P Brand Tri-Angle, 50 ml. Baxter B2722-50A). - Fill molds with complete Epon mix and put samples into mold or Beem capsules.
- Place in oven at 600C for 2 days.