Plant and Animal Microtechnique using Microwave Energy

Protocol

Authors

Steven Ruzin, Denise Schichnes

Overview

These are general protocols for paraffin microtechnique using specialized microwave ovens. When performing the procedures described microwave energy greatly accelerates the process.

Background

The traditional method of preparing microscope tissue preparations involves a transferring the tissues through a series of solutions (fixative, dehydrating, liquid paraffin) over a lengthy period of time, usually from one to two weeks. The time can be decreased greatly by using a specialized Tissue Processing Microwave Oven to accelerate all steps of the procedure. Through the use of MW energy tissue can be processed and ready for sectioning in one to two days. In many cases the use of MW energy can also increase the quality of tissue preservation.

Materials/Equipment

Tissue processing oven: We recommend the MRA BP111RS tissue processing microwave oven because of its microwave uniformity and precise MW power control. Other acceptable ovens are made by Ted Pella, EBS, and EMS.

Fixatives: Formaldehyde

Dehydrating agent: Isopropanol

Embedding medium: Paraffin blend for Microtechnique. The standard companies will work fine.

Units, terms, definitions

Formalin: 37-40% formaldehyde in stabilized water

FAA: Formalin, Acetic Acid, Alcohol (EtOH)

FPA: Formalin, Propionic Acid, Alcohol

Sample Bath: plastic or glass pan with samples in holder. The pan is filled with water to the level of the liquid in the sample vials.

MW: Microwave Tissue Processing Oven. This is a specialized oven designed for processing histological samples. A commercial or consumer MW oven will not work with this protocol

MW Power: This is the % Full Power of the MW Oven.

RT: Room temperature

EtOH: Ethanol

Procedure

Procedure for Animal tissue. For all steps of the procedure the MW power should be 750W.

  1. Microwave Fix tissues in 10% formalin. MW for 30min at 37C. Sample bath temp = 4-6∘C.
  2. Transfer to 50% EtOH. MW for 30min at 67∘C. Sample bath at RT
  3. Transfer to 95% EtOH. MW for 30min at 67∘C. Sample bath at RT
  4. Transfer to 100% EtOH + 1% (w/v) Eosin Y. MW for 10min at 67∘C. Sample bath at RT
  5. Transfer to 1:1 Abs EtOH:Isopropanol. MW for 30min at 77∘C. Sample bath at RT
  6. Transfer to 100% Isopropanol. MW for 30min at 77∘C. Sample bath at RT
  7. Transfer to 1:1 Isopropanol:liquid paraffin. MW for 30min at 77∘C. Sample bath at 60∘C
  8. Transfer to liquid paraffin. MW for 90min at 67∘C. Sample bath at 60∘C
  9. Transfer to liquid paraffin. MW for 90min at 67∘C. Sample bath at 60∘C
  10. Embed tissues immediately.
  11. Section at 7μm, mount to glass microscope slides, and stain according to published protocols.

Procedure for Plant tissue. For all steps of the procedure the MW power should be 650W.

  1. Microwave Fix tissues in FAA or FPA 15min at 37∘C. Sample bath 4C.
  2. Transfer to fresh FAA or FPS and microwave fix tissues 15min at 37∘C. Sample bath 4∘C
  3. Transfer to fresh FAA or FPS and microwave fix tissues 15min at 37∘C. Sample bath 4∘C
  4. Transfer to 70% EtOH, MW 5min at 67∘C. Sample bath RT
  5. Transfer to 85% EtOH, MW 5min at 67∘C. Sample bath RT
  6. Transfer to 95% EtOH, MW 5min at 67∘C. Sample bath RT
  7. Transfer to 100% EtOH + 1% w/v Safranin O, MW 5min at 67∘C. Sample bath RT
  8. Transfer to 100% EtOH + 1% w/v Safranin O, MW 5min at 67∘C. Sample bath RT
  9. Transfer to 1:1 EtOH:Isopropanol, MW 5min at 77∘C. Sample bath RT
  10. Transfer to 100% Isopropanol, MW 5min at 77∘C. Sample bath RT
  11. Transfer to 1:1 Isopropanol:liquid paraffin. MW 10min at 77∘C. Sample bath RT
  12. Transfer to 100% liquid paraffin. Sample bath 60∘C
  13. Transfer to 100% liquid paraffin. Sample bath 60∘C
  14. Transfer to 100% liquid paraffin. Sample bath 60∘C
  15. Transfer to 100% liquid paraffin. Sample bath 60∘C
  16. Transfer to 100% liquid paraffin. Sample bath 60∘C
  17. Embed tissues immediately
  18. Section at 7-10μm, mount to glass microscope slides and stain according to published protocols.

Notes and troubleshooting tips

  1. When changing solutions, transfer the liquid and not the tissues. This minimizes tissue damage.
  2. If the paraffin solidifies between paraffin steps of the protocol, place the tissue vials into a paraffin oven and allow the paraffin to completely liquefy before resuming the steps.

Links to resources and suppliers

Microwave Research & Applications: http://www.microwaveresearch.com/

Ted Pella: http://www.tedpella.com/

Electron Microscopy Sciences: http://www.emsdiasum.com/

Energy Beam Sciences: http://www.ebsstore.com/

Literature references

Microwave Protocols for Plant and Animal Paraffin Microtechnique. Denise Schichnes, Jeffrey A. Nemson, and Steven E. Ruzin. Microscopy Today. May 2005.

Giberson, R.T., R.S. Demaree Jr. eds, Microwave Techniques and Protocols. Humana Press Inc, Totowa, New Jersey.

Johansen, D.A. (1940) Plant Microtechnique. New York: McGraw-Hill Book Company, Inc.

Kok L.P., P.E. Visser, M.E. Boon (1988) Histoprocessing with the Microwave Oven: an Update. Histological Journal 20:323-328.

Ruzin S.E. (1999) Plant Microtechnique and Microscopy. Oxford University Press, Cambridge.

Schichnes D., J.A. Nemson, S.E. Ruzin (2001) Microwave Paraffin Techniques for Botanical Tissues. In Giberson R.T., Demaree R.S. eds, Microwave Techniques and Protocols. Humana Press Inc, Totowa, New Jersey, pp 181-189.

Health, safety & hazardous waste disposal considerations

Formaldehyde is a known carcinogen and a California Prop 65 chemical. It must be disposed of according to local and Federal regulation.

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