EM Processing Protocol

Microwave Used: PELCO BioWave® Pro - Protocol 1

     
Microwave Configuration AMicrowave Configuration BMicrowave Configuration C
                                                                                                             
Step WattageMW Chamber
 Configuration
TimeReagent NotesProcess Notes
Fixation
 Glutaraldehyde
1 step<200WA1 off - 1 on - 3 offStart with fixative
 between 4 and 20°C
Time in Minutes
 Vacuum 20" Hg
Fixation
Karnovsky's or
 Formaldehyde*
Step 1150WA1 on - 1 off - 1 onStart with fixative
 between 4 and 20°C
Time In Minutes
 Wattage change
Step 2650WA10 on - 20 off - 10 onTime in Seconds
 Vacuum 20" Hg
*NOTE: Formaldehyde containing fixatives need a two-step fixation process for fresh tissue
Fixation
 Osmium**
Step 1<100WA2 on - 2 off - 2 onStart with fixative
 between 4 and 20°C
Time in Minutes
Step 2<100WA2 on - 2 off - 2 onCool fixative if T>27°C
 before starting Step 2
Time in Minutes
**NOTE: Osmium penetration into tissue is best at low wattage
Dehydration
50% ETOHStep 1<250WB40 secStart with ETOH or acetone
 at room temperature
70% ETOHStep 2<250W40 sec
90% ETOHStep 3<250W40 sec
100% ETOHStep 4<250W40 sec
100% ETOHStep 5<250W40 sec
NOTE: the same steps can be used for acetone in place of ETOH
Resin Infiltration
1:1 acetone:resinStep 1>200WA3-5 minResin viscosity will determine
 wattage and time
Vacuum 20" Hg
100% resinStep 2>200W3-5 min
100% resinStep 3>200W3-5 min
Spurr's & LR White 200W - Epon 250W - Araldite 300W Use longer times based on sample and resin
Resin Polymerization
EpoxyStep 1150WC30 minThe first 30 minutes of
heating should not
 exceed 60°C for best results
Elevate dish above
ColdSpot with
 vacuum glass ring
EpoxyStep 2750W60 min
LR WhiteStep 1150W30 min
LR WhiteStep 2750W30 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.