MHP Protocol

Created by Karen Darley, Modified on Mon, 27 May 2019 at 01:34 PM by Ben Peterson

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
Fixation Karnovsky's or Formaldehyde*	Step 2	650W	A	10 on - 20 off - 10 on	Start with fixative between 4 and 20°C	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
Fixation Osmium**	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.