EM Processing Protocol
Microwave Used: PELCO BioWave® Pro - Protocol 1
 |  |  |
| Microwave Configuration A | Microwave Configuration B | Microwave Configuration C |
| Step | Wattage | MW Chamber Configuration | Time | Reagent Notes | Process Notes | |
| Fixation Glutaraldehyde | 1 step | <200W | A | 1 off - 1 on - 3 off | Start with fixative between 4 and 20°C | Time in Minutes Vacuum 20" Hg |
| Fixation Karnovsky's or Formaldehyde* | Step 1 | 150W | A | 1 on - 1 off - 1 on | Start with fixative between 4 and 20°C | Time In Minutes Wattage change |
| Step 2 | 650W | A | 10 on - 20 off - 10 on | Time in Seconds Vacuum 20" Hg | ||
| *NOTE: Formaldehyde containing fixatives need a two-step fixation process for fresh tissue | ||||||
| Fixation Osmium** | Step 1 | <100W | A | 2 on - 2 off - 2 on | Start with fixative between 4 and 20°C | Time in Minutes |
| Step 2 | <100W | A | 2 on - 2 off - 2 on | Cool fixative if T>27°C before starting Step 2 | Time in Minutes | |
| **NOTE: Osmium penetration into tissue is best at low wattage | ||||||
| Dehydration | ||||||
| 50% ETOH | Step 1 | <250W | B | 40 sec | Start with ETOH or acetone at room temperature | |
| 70% ETOH | Step 2 | <250W | 40 sec | |||
| 90% ETOH | Step 3 | <250W | 40 sec | |||
| 100% ETOH | Step 4 | <250W | 40 sec | |||
| 100% ETOH | Step 5 | <250W | 40 sec | |||
| NOTE: the same steps can be used for acetone in place of ETOH | ||||||
| Resin Infiltration | ||||||
| 1:1 acetone:resin | Step 1 | >200W | A | 3-5 min | Resin viscosity will determine wattage and time | Vacuum 20" Hg |
| 100% resin | Step 2 | >200W | 3-5 min | |||
| 100% resin | Step 3 | >200W | 3-5 min | |||
| Spurr's & LR White 200W - Epon 250W - Araldite 300W Use longer times based on sample and resin | ||||||
| Resin Polymerization | ||||||
| Epoxy | Step 1 | 150W | C | 30 min | The first 30 minutes of heating should not exceed 60°C for best results | Elevate dish above ColdSpot with vacuum glass ring |
| Epoxy | Step 2 | 750W | 60 min | |||
| LR White | Step 1 | 150W | 30 min | |||
| LR White | Step 2 | 750W | 30 min | |||
| (1) Use temperature probe to check solution temperature after the microwave step is completed. | ||||||
| The temperature probe will act as a microwave antenna in small fluid volumes creating a sample temperature profile that would not exist otherwise. | ||||||