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NEW PELCO BioWave® Pro+ Microwave Tissue Processor
go to the Electron Microscopy Tissue Processing Application Kit

Microwave Protocols for Electron Microscopy


Submitted by RONALD L. AUSTIN
Dept. of Pathology, LSUHSC, Shreveport, LA

Microwave Processing in a Clinical Lab
Daily Routine - Microwave Protocol

Microwave Setup and Processing Protocol for Electron Microscopy

Total processing time: ~30 minutes - Resin Polymerization: 45-75 minutes

electron microscopy specimen preparation in the biowave pro processor using a vacuum chamber on the pelco coldspot microwave processing configuration on the pelco coldspot microwave processing for electron microscopy using the pelco biowave pro

Fixation - Resin Infiltration
Microwave Configuration A

PELCO® EM Pro Vacuum Chamber with
PELCO Prep-Eze™ Specimen Holder inside
on the PELCO ColdSpot® Pro

Dehydration Steps
Microwave Configuration B
PELCO Prep-Eze™ Specimen Holder
on the PELCO ColdSpot
® Pro

Resin Polymerization
Microwave Configuration C

Microwave Polymerization System shown with
PELCO® Microwave Capsule Holder,
Polymerization Stand and PELCO ColdSpot® Pro


Step   Wattage MW Chamber
Configuration
Time Reagent Notes Process Notes
Fixation
Glutaraldehyde
1 step 150W 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 sec on - 20 sec off - 10 sec 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 -2 off - 2 on Start with fixative
at room temperature
Time in Minutes
Step 2 100W A 2 on - 2 off - 2 on x2 Cool fixative if T>27°C
before repeating
Time in Minutes
**NOTE: Osmium penetration into tissue is best at low wattage
Dehydration
50% ETOH Step 1 150W B 40 sec Start with ETOH or acetone
at room temperature
70% ETOH Step 2 150W 40 sec
90% ETOH Step 3 150W 40 sec
100% ETOH Step 4 150W 40 sec
100% ETOH Step 5 150W 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
Epoxy Step 2 450W 45 min
LR White Step 1 150W 30 min
LR White Step 2 450W 15 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.

The protocol above is based on:

Giberson, R.T., Austin, R.L., Charlesworth, J., Adamson, G., Herrera, G.A. (2003) Microwave and digital imaging technology reduce turnaround times for diagnostic electron microscopy. Ultrastruct. Pathol. 27:187-196.

Microwave-assisted tissue processing for electron microscopy is no different from conventional processing methods. The reagents used on the bench are typically used in the microwave. The number of steps in conventional processing are usually converted into microwave steps.

Microwave-assisted Fixation:

The microwave steps for fixation are programmed differently for glutaraldehyde than for fixation with formaldehyde or formaldehyde containing fixatives such as Karnovsky's (see appendix). Recent work (see below) has demonstrated the need for two different wattage settings when formaldehyde is used alone or as part of a mixed fixative.

Galvez, J.J., Giberson, R.T., Cardiff, R.D. (2006) The role of microwave radiation in reducing formaldehyde fixation times. The J. Histotechnol. 29:113-121.

The specimen holder routinely used for microwave processing is the PELCO Prep-Eze™ Kit (36157, Fig. 1). There is also a microcentrifuge tube holder (36134, Fig. 1) supplied with the EM Kit. The Prep-Eze™ is placed in 58x15mm polypropylene petri dishes (36135) which are half- filled with reagent (Figs. 2 & 3).

Microwave Fixation Fig. 1 Microwave Fixation Fig. 1A NOTE: More than one Prep-Eze can be processed at one time.They can also be processed side-by-side with the microcentrifuge tube holder.
The microcentrifuge tube holder can be used for sample fixation and processing as well as the Prep-Eze. The arrow indicates the approximate level to fill the tube for processing. The Prep-Eze, designed for bench or microwave sample processing for electron microscopy, is available as a 6-well (#36157) and 12-well (#36158) kit. The ID Mats shown above can be written on with a Sharpie and the markings removed by wetting a towel with acetone and wiping the surface. Taped to the counter they serve as an easy-to-use method of specimen identification.

The Prep-Eze™ is placed in the vacuum chamber for aldehyde and osmium fixation, en bloc staining and resin infiltration (Figs. 4). The vacuum chamber is then placed on the ColdSpot as shown in Figure 5. Fixation by Microwave Fig 3
The PELCO BioWave® Pro is programmed with individual protocols for fixation with glutaraldehyde, osmium, ethanol or acetone dehydration, resin infiltration and resin polymerization (epoxies) as separate protocols.  There is also the complete processing protocol incorporating all steps mentioned above.  Protocol steps can be edited to suit your processing requirements.  Dehydration steps are done with the Prep-Eze™ on the ColdSpot as shown in Figure 6.  Microwave Fixation 5 Microwave Fixation 5

Resin polymerization requires placing the embedding capsules in the PELCO® Microwave Capsule Holder with Lid (36131-2, see right) and then placing the holder into the Microwave Polymerization Tray (36133, Figs. 7-10, below - right).
The capsule holder is designed for 00 embedding capsules, either conical tip or flat bottomed. The Capsule Preparation Station (see below) is designed for filling the embedding capsules with resin and to keep the Microwave Capsule Holder free of any spilled resin during that process.

Capsuel Preparation SationCapsule Preparation Station

   
Microwave Capsule Holder
Un-assembled Assembled
Figures 7-10 demonstrate the setup for resin polymerization in the microwave.

Appendix:

1. Fixation steps with paraformaldehyde or Karnovsky's fixative - Protocol
Step# Description User Prompt
(on/off)
Time
(hr:min:sec)
Power
(Watts)
Temp.
(°C)
Load Cooler
(off/auto/on)
Vacuum/
Bubble Pump
(off/bub/
vac cycle/
vac on)
1 Karnovsky's Fixation On On 0:1:0 150 50 Auto Vacuum cycle
2 Karnovsky's Fixation Off Off 0:3:0 0 50 Auto Vacuum cycle
3 Karnovsky's Fixation On Off 0:1:0 150 50 Auto Vacuum cycle
4 Karnovsky's Fixation Off Off 0:2:0 0 50 Auto Vacuum cycle
5 Karnovsky's Fixation On Off 0:0:10 650 50 Auto Vacuum cycle
6 Karnovsky's Fixation Off Off 0:0:20 0 50 Auto Vacuum cycle
7 Karnovsky's Fixation On Off 0:0:10 650 50 Auto Vacuum cycle
8 Buffer Rinse On 0:0:40 150 50 Auto Off
9 Buffer Rinse On 0:0:40 150 50 Auto Off
10 Osmium On On 0:2:0 100 50 Auto Vacuum cycle
11 Osmium Off Off 0:2:0 0 50 Auto Vacuum cycle
12 Osmium On On 0:2:0 100 50 Auto Vacuum cycle
13 Osmium Off Off 0:2:0 0 50 Auto Vacuum cycle
14 Osmium On On 0:2:0 100 50 Auto Vacuum cycle
15 Water Rinse On 0:0:40 150 50 Auto Off

Notes:
Vacuum Cycle = Vacuum and Vent
For Continuous Vacuum set vent time to zero


2. En bloc staining steps after osmium fixation - Protocol
Step# Description User Prompt
(on/off)
Time
(hr:min:sec)
Power
(Watts)
Temp.
(°C)
Load Cooler
(off/auto/on)
Vacuum/
Bubble Pump
(off/bub/
vac cycle/
vac on)
1 Uranyl Acetate On On 0:1:0 100 50 Off Vacuum cycle
2 Uranyl Acetate On Off 0:1:0 0 50 Off Vacuum cycle
3 Uranyl Acetate On Off 0:1:0 100 50 Off Vacuum cycle

Notes:
Vacuum Cycle = Vacuum and Vent
For Continuous Vacuum set vent time to zero




Fig. 1A-C. Figure 1A is a micrograph of a normal sural nerve with a non-myelinated nerve (N) having secretory vesicles (sv), microtubules (mt) and a swan cell nucleus (ScN). The insert (1B) shows a myelinated nerve and the arrows clearly demonstrate its periodicity. Figure 4C is a membranouse Lupus nehpritis (RPS/ISN Class V). There is diffuse, generalized effacement of the foot processes of the visceral epithelial cells. Numerous regularly disposed epimembraneous immune complex deposits are illustrated by the arrows. Both tissues were initially fixed in a variant of 10% NBF (Carson et al. 1972) and then processed in the microwave for ultrastructural evaluation by the methods of Giberson et al. (2003) and Austin (2002). Micrographs from Ronald L. Austin, Research Associate, Dept. of Pathology, LSU Medical Center, Shreveport, LA 71130.




Figure 2A-G. Fig. 2A-B shows cytoplasmic iridovirus from the skin of a sturgeon. The iridovirus is a large enveloped dsDNA virus which infects both insect and vertebrate hosts. Fig. 2C-E demonstrates an intranuclear baculovirus from the hepatopancreas of a crayfish from Northern California. Fig. 2C is a low magnification image of the enveloped dsDNA virus showing the intranuclear arrangement of virus particles Fig. 2D is a higher magnification showing both a cross-sectional and longitudinal view of the virus. Fig. 2E is a high magnification cross-section of a number of virus particles demonstrating the unique intranuclear membrane-bound virions. Fig. 2F-G demonstrates an endothelial cell polyoma virus from a blood vessel in the liver of a parakeet. Polyoma virus have a single molecule of circular dsDNA and the particles are non-enveloped. Polyoma virions are spherical in outline and typically 45nm in diameter. Fig. 2F is a low magnification image showing typical nuclear presentation. Fig. 2G is a high magnification view of the virus. Infected tissues were processed directly from 10% NBF by the microwave methods of Nordhausen and Barr (2001) and Nordhausen et al. (2002). Micrographs from Bob Nordhausen, Univ. of California, Davis, California Animal Health and Food Safety Lab, School of Veterinary Medicine, Davis, CA 95616.




Figure 3. Micrographs from a 2008 microwave workshop held at the Center for Microscopy, San Joaquin Delta College, Stockton, CA. Rat brain (not perfusion fixed) (1), cardiac muscle (2), kidney (3) and liver (4) were processed from osmium through resin polymerization for a net turnaround time of under 4 hours from fresh tissue to the electron microscope. Microwave techniques (Giberson, et al., 2003) make it possible to teach and in real time demonstrate the techniques of electron microscopy.

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