PELCO BioWave® Pro+ Microwave Tissue Processor
Electron Microscopy Tissue Processing Application Kit

Microwave Protocols for Electron Microscopy

Microwave Setup and Processing Protocol for Electron Microscopy

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

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

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).

		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.

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.

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. Capsule Preparation Station
	Un-assembled	Assembled

Figures 7-10 demonstrate the setup for resin polymerization in the microwave.

Appendix:

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.