EQU - Laboratory Vacuum Microwave Processing

Introduction:

Microwave technology has reduced the times required for sample processing by over 90%, when compared to routine processing protocols. Water recirculation, the temperature probe and variable wattage have made protocol development possible. The addition of a microwave vacuum chamber has improved sample preservation and reduced times required for resin infiltration by >80%, over previously established microwave protocols. (Giberson et al., 1997, J. Vet. Diag. Invest. 9:61-67)

Protocol:1. Microwave Oven Calibration

Figure 1 indicates the placement of the finder grid mat, water loads and the vacuum chamber during VMP. The combination of water loads shown in Figure 1 creates an excellent cold spot during the fixation and buffer rinse steps. The heating rate for the microcentrifuge tubes should be 8-10°C (when 600µl of water is used - 40 seconds at 100% power under vacuum - 20"Hg).

It is best to determine the actual heating rate with the buffer and fixative combination being used. Osmium will tend to heat less than the aldehydes over the same time interval.

Fig. 1. The finder mat grid is taped to the oven cavity, approximately 1" from the front of the microwave cavity and centred side to side. An 800ml or 1 L plastic beaker (glass is all right but it creates more heat in the oven cavity) is filled with 500-700ml of tap water and the water is recirculated and cooled to under 35°C using the Load Cooler. The vacuum chamber is placed, as shown, in the microwave cavity. It has 2 each 100ml water loads inside which are always oriented as shown. The holder for the sample tubes (A) is placed in front to the two water loads, so that one of the sample tubes is directly below the temperature probe, which is inserted through the O-ring in the top of the chamber (see Fig. 3).

Protocol 2. Aldehyde Fixation:

Microcentrifuge tubes which contain the specimens and 600µl of fixative are placed in the microcentrifuge tube holder (Fig. 2) which is positioned in the vacuum chamber as shown in Fig.1. A vacuum (20" Hg; 500 torr) is drawn. The microwave is then programmed for the following time sequences (these three time/power intervals are programmed sequentially on one numbered key pad):

1. 1 minute at 0% power
2. 40 seconds at 100% power
3. 3 minutes at 0% power

The starting temperature of the fixative should be ≤20°C.

Fig. 2 The microcentrifuge tube holder is made of PTFE. The volume of fixative in the microcentrifuge tube should be 600µl ±100µl. Variations greater than 100µl will change the heating rate of the fixative solution.

Figure 3 is a side view of the vacuum chamber. The temperature probe is to be positioned about 3mm above the surface of the fixative in one of the sample tubes. At the end of the 40 seconds at 100% power the microwave door is opened and the temperature probe is pushed down into the fixative to record the temperature after microwave irradiation. The temperature change should be >8°C. After noting the temperature, close the microwave door and push the start button. The final 3 minutes of 0% power will count down.

Fig. 3. The temperature probe (C) is positioned ~3mm above the fixative level in one of the microcentrifuge tubes. Beaker "A" is a standard disposable 100ml polypropylene beaker. The 3-way vacuum valve (D) is positioned toward the front of the microwave, the vacuum hose attached and a vacuum drawn (20" Hg; 500 torr). Close the valve. When the temperature probe is pushed down into the fixative to record the final temperature, after microwave irradiation, the vacuum will remain intact.

Protocol 3. Buffer Rinse:

At the end of the 3 minute 0% power sequence, break the vacuum and remove the holder and microcentrifuge tubes. Remove the fixative and replace with buffer. Immediately remove the buffer and add 600µl of fresh buffer. Place the holder with tubes back in the vacuum chamber, draw a vacuum (20" Hg; 500 torr) and microwave for the following intervals:

1. 1 minute at 0% power
2. 40 seconds at 100% power

Protocol 4. Osmium Fixation:

Remove the buffer and add 600µl of osmium fixative. It is best if the fixative temperature is <20°C before starting. Repeat the steps for aldehyde fixation (1 min. 0% power / 40 Sec. 100% power / 3 min. 0% power). At the end of the sequence, remove the osmium from the samples (under the fume hood) and rinse the tissue with water. Make sure to note the temperature after the 100% power cycle. In our experience the osmium step will heat less than the aldehyde under vacuum.

NOTE:

Replace the two 100ml water loads with fresh tap water prior to starting osmium fixation.

Protocol 5. Water Rinse:

Quickly rinse the samples with tap water prior to transferring them to the flow-through baskets for dehydration (see Giberson, et al., 1997) and resin infiltration.

Protocol 6. Dehydration:

We typically use acetone in the following concentrations: 1 x 50%; 1 x 70%; 1 x 90%; 2 x 100%. Ethanol can be used in place of acetone if desired. A temperature restriction (37°C) is used during the dehydration steps which entail 40 seconds at 100% power for each step. The flow-through baskets are placed in polypropylene petri dishes and approximately 15-18ml of solution is used for each step (Fig. 4).

The Petri dish with baskets, shown in figure 4, is placed in front of the water load (about 4") serviced by the load cooler and a second water load of about 400ml is place to the right of the petri dish. The temperature probe is placed in the probe stand.

Protocol 7. Vacuum Resin Infiltration:

The second water load, added for dehydration, is removed as are the two 100ml water loads from the vacuum chamber. The petri dish with baskets is placed in the vacuum chamber. The lid to the vacuum chamber, with the temperature probe inserted, is placed on the top of the chamber (Fig. 3). Make sure the tip of the temperature probe is in the resin in the petri dish. Position the vacuum chamber in the same location that was used for fixation. Set the temperature restriction to 43°C and draw a vacuum (20" Hg; 500 torr). Three two minute vacuum infiltration steps are done in the microwave (100% resin is used for each infiltration step). Use fresh resin for each step. After the last two minute run the tissue is ready for embedding in capsules and polymerisation.

Protocol 8. Polymerisation:

The embedding capsules are polymerised under water in the microwave (see Giberson, et al., 1997) (Fig. 5). Since the publication of Giberson, et al., 1997, we have found that the use of the temperature probe during polymerisation improves the shape and overall block quality after polymerization.

In figure 5 a 1000ml, rectangular polypropylene dish is used during polymerisation. The large water load (serviced by the load cooler is left in the microwave. Water should be added as needed to maintain a level above that of the embedding capsules.

The following polymerisation schedules are recommended for the resins listed:

• Epoxies: 10 min. at 60°C; 10 min. at 70°C; 10 min. at 80°C; 45 min. at 100°C
• LR White: 10 min. at 60°C; 10 min. at 70°C; 25 min. at 80°C
• Histocryl: 10 min. at 60°C; 10 min. at 70°C; 10 min. at 80°C; ~30 min. at 90°C

The Effect of Microwaves/Vacuum on the Sample Processing Times for Electron Microscopy

Microwave‐assisted processing of human colon endoscopic biopsy after normal 10% NFB fix.

1 x 7 minutes 100% Ethanol @ 350W TR of 67°C

1 x 5 minutes ACS reagent Grade Isopropanol @ 350W, TR of 74°C1 x 18 minutes Paraffin @ 650W, TR of 80°C (400x approximate) H&E.

Liver stained with Masson's Trichrome stain using the PELCO HistoWave® (20x). Courtesy of Rick Giberson, Ted Pella, Inc. and Bruce Calkins, Pathology Sciences, Chico.

Intestine, cut at 4 microns, stained with Mucin Alcian Blue pH2.5 using the PELCO HistoWave® (20x). Courtesy of Rick Giberson, Ted Pella, Inc. and Bruce Calkins, Pathology Sciences, Chico.

Intestinal cross‐section, cut at 4µm, stained with PAS using the PELCO HistoWave® (20x). Courtesy of Rick Giberson, Ted Pella, Inc. and Bruce Calkins, Pathology Sciences, Chico.

Microwave Calibration

The Microwave Calibration Slide Set is a set of glass slides with liquid crystal temperature strips permanently affixed to them. The slides help you predict the temperature of the staining solution around a tissue section during and after microwave irradiation. You will have two sets of calibration slides for microwave staining. Those labelled Calibration Slide #1 are for low‐temperature staining (35°C to 45°C). Those labelled Calibration Slide #2 are for high‐temperature staining (50°C to 60°C).

PRECAUTION:

• Do not heat solutions containing the Calibration Slides above 65°C. The warm solution will melt the adhesive on the LCT strip, and the LCT strip will fall off the slides.
• To avoid pressure build‐up in the glass or plastic staining jars, do not cover the staining jars.
• If your staining protocol calls for bringing the solution to a boil or for steam at pressure, use a microwave pressure cooker. The unit is designed to safely handle 10lb/in² within a few minutes of heating in a microwave oven. Do not use your Calibration slide Set in the pressure cooker: the high temperature will melt the adhesive on the LCT strip.
• Do not irradiate the neon bulbs longer than 1 minute. They will become too hot to handle and could be damaged. Let the Neon Bulb Array cool for 2 minutes before re‐irradiation.

Procedure The Calibrating Your Oven For Batch Microwave Staining:

This procedure will allow you to predict, with confidence, the temperature of the staining solution around the microwave‐irradiated tissue sections.

Materials Needed:

• Distilled water, 150ml
• 3 plastic staining jars, 30ml size.
• Notebook.
• Red marker.
• Thermal mitts.
• Microwave finder map, or alpha‐numeric oven tray.
• Neon Bulb Array and diagram of Neon Bulb Array.
• Microwave Calibration Slide Set.
• Microwave Tool Book.

Calibrating Procedure:

1. Make sure the alpha‐numeric Grid is in the left, rear corner of the microwave oven.
2. Warm up the oven electronics by placing a beaker filled with 250mL of water in the right, rear corner of the oven. Program the oven to irradiate for 2 minutes at 100% power. Press start. Begin the next step within 2 minutes after the oven shuts off, or you must repeat this step.
3. Place the Neon Bulb Array on top of the alpha‐numeric Grid. Align it so the corner with the large dot is at the left rear of the alpha‐numeric Grid and oven.
4. Close the oven door. Programme the oven to irradiate for 30 seconds at 100% power. Press start.
5. Observe the neon bulbs. Look for clusters of three to four bulbs that are continuously lit. Mark the alpha‐numeric Grid co‐ordinates corresponding to these bulbs on the diagram of your Neon Bulb Array.
6. When the oven turns off, let the bulbs cool for 2 minutes. Meanwhile, fill a staining jar with 50ml or room‐temperature distilled water.
7. Remove enough bulbs from one cluster that was lit so that you can place the staining jar between the bulbs at that co‐ordinate.
8. Close the oven door. Programme the oven to irradiate at 100% power for 20 seconds. Press start.
9. See if bulbs around the staining jar are continuously lit. If bulbs around the jar do not light up, refill the staining jar with 50ml of room temperature distilled water and reposition the jar on the Neon Bulb Array at another maximum power cluster (from step v.) Repeat steps 2 to 9 until you have identified a position where the bulbs remain continuously lit around the staining jar. Record your observations in your notebook.
10. With the red marker, mark the alpha‐numeric grid at the location identified in step 9, drawing a red circle around the base of the jar. This is the optimal place to put samples for staining procedures. It is an area of maximum power with your loaded staining jar.
11. Remove the Neon Bulb Array.
12. Refill the staining jar with 50ml of room temperature distilled water and place it on the area marked in red on the alpha‐numeric grid.
13. Place one calibration slide (from set #1 or set #2, as appropriate for your staining protocol) in the centre slot of the staining jar.
14. Programme the oven to irradiate at 100% power for 5 seconds. Press start.
15. As soon as the oven shuts off, open the oven door and observe the colour of the liquid crystals on the slide.
16. Repeat steps 12 to 15 with the same slide, but adjust the irradiation time until the liquid crystal turns bright green for the target temperature you will use in your staining protocol.
17. In your notebook, record the optimal alpha‐numeric grid co‐ordinates, irradiation time, and temperature for the calibration slides in the staining jar.
18. Optional step: If your protocol requires simultaneous use of two to three staining jars, repeat steps 5 to 17 using three staining jars, each filled with 50ml water.
    

NOTE: Some ovens do not produce enough power to reproducibly heat three jars at once.

Procedure for standardised protocol for batch microwave staining:

Using a standardised protocol for each microwave staining series improves reproducibility. We recommend using this protocol as a guide for adapting published staining protocols for your microwave oven.

Batch Microwave Staining Procedure:

1. Warm up the oven electronics for 2 minutes.
2. Use the neon bulb array to identify the best location in the oven for microwave staining.
3. Use alpha‐numeric grid for reproducible placement of the staining jars in the oven.
4. Use calibration slides to check irradiation conditions in the loaded microwave cavity.
5. Use standardised staining jars.
6. Be sure all surfaces in the oven are dry
7. Make sure the alpha‐numeric grid is in the left, rear corner of the oven.
8. Warm up the oven electronics by placing the beaker with 250ml of water in the right, rear corner of the oven. Programme to oven to irradiate for 2 minutes at 100% power. Press start. Begin the next step with 2 minutes after the oven shuts off, or repeat this step.
9. Select the calibration slide set that corresponds to the final temperature range you want to achieve. Place the slide in your staining jar.
10. Place 50ml of the solution you will use for staining into the staining jar.
11. Place the staining jar(s) on the alpha‐numeric grid on the co‐ordinated recorded from the result 17 above.
12. Programme the oven for the power and time conditions described in your microwave staining protocol. Press start. After the microwave oven stops, the calibration slides should show the expected endpoint temperature for the staining protocol. If they do not, check the following conditions against your calibration procedure (above): staining jar location on the alpha‐numeric grid, staining solution type, volume, initial temperature, and selected irradiation time and power settings.
13. Immerse slides containing tissue sections in 50ml of fresh, room‐temperature staining solution in the staining jar(s).
14. Irradiate the tissue sections at the alpha‐numeric grid co‐ordinated for the power and time condition that resulted in the most even heating of the calibration slide set (from step 7). Press start.
15. Complete the staining protocol by following the published procedure of your choice.

Reference:

The Microwave Tool Book ‐ A Practical Guide for Microscopists. Login and Dvorak. Beth Israel Hospital Boston. ISBN 0‐9642675‐0‐0