Structural and antigenic preservation of plant samples by microwave-enhanced fixation, using dedicated hardware, minimizing heat-related effects. 2004

Francisca Lería, and Roberto Marco, and Francisco Javier Medina
Centro de Investigaciones Biológicas (CSIC), E-28040 Madrid, Spain.

We explored the use of microwave technology in fixation with the objective of achieving quicker fixation regimes, lower concentrations of toxic and volatile reagents, and enhanced antigen detection. We used a modified domestic microwave oven (900 W) and a low-power (5 W) microwave bench. The work was done on plant materials. The oven was supplemented with a cooling device, a stirring system, and a record of the sample temperature and the time of effective irradiation. The sample, immersed in a fixative solution of 1% paraformaldehyde (PFA) in PBS, was irradiated for only 10 minutes. The sample temperature did not exceed 37 degrees C. In these mild conditions, the quality of the (ultra)structural preservation of the samples, morphometrically assessed, was at the same level as obtained with the same fixative, using conventional methods. On the contrary, samples fixed in the same conditions without irradiation showed a poor structural preservation. The antigenic preservation of the irradiated samples was excellent, since the labeling levels of two nucleolar proteins, detected by immunogold, were three times higher than in conventionally fixed samples. In the so-called microwave bench, the pathway of microwaves is guided, so that low-power microwaves directly hit the sample and there is no dispersion of energy. Temperature of fixative did not increase after microwave irradiation. Fixation in the bench with either 4% PFA, or 1% PFA, for 20 minutes resulted in structural preservation of samples similar in quality as obtained with conventional fixation and in a similar or better level of antigen preservation. Therefore, controlling temperature and effective irradiation is crucial in order to obtain optimal structural and antigen preservation with microwave-enhanced fixation. The dramatic differences observed between microwave-irradiated samples and samples fixed in the same conditions without irradiation, strongly support the existence of specific effects of microwaves on fixation, independent from the mere heating of the samples.

UI MeSH Term Description Entries
D008872 Microwaves That portion of the electromagnetic spectrum from the UHF (ultrahigh frequency) radio waves and extending into the INFRARED RAYS frequencies. EHF Waves,Extremely High Frequency Radio Waves,Micro Wave,Micro Waves,Ultrahigh Frequency Waves,Microwave Radiation,EHF Wave,Micro Waves,Microwave,Microwave Radiations,Radiation, Microwave,Ultrahigh Frequency Wave,Wave, EHF,Wave, Micro,Wave, Ultrahigh Frequency,Waves, Micro
D011108 Polymers Compounds formed by the joining of smaller, usually repeating, units linked by covalent bonds. These compounds often form large macromolecules (e.g., BIOPOLYMERS; PLASTICS). Polymer
D005404 Fixatives Agents employed in the preparation of histologic or pathologic specimens for the purpose of maintaining the existing form and structure of all of the constituent elements. Great numbers of different agents are used; some are also decalcifying and hardening agents. They must quickly kill and coagulate living tissue. Fixative,Pickling Agents,Agents, Pickling
D005557 Formaldehyde A highly reactive aldehyde gas formed by oxidation or incomplete combustion of hydrocarbons. In solution, it has a wide range of uses: in the manufacture of resins and textiles, as a disinfectant, and as a laboratory fixative or preservative. Formaldehyde solution (formalin) is considered a hazardous compound, and its vapor toxic. (From Reynolds, Martindale The Extra Pharmacopoeia, 30th ed, p717) Formalin,Formol,Methanal,Oxomethane
D000490 Allium A genus of the plant family Amaryllidaceae. Many produce pungent, often bacteriostatic and physiologically active compounds and are used as VEGETABLES; CONDIMENTS; and medicament, the latter in traditional medicine. Alliaceae
D000941 Antigens Substances that are recognized by the immune system and induce an immune reaction. Antigen
D016707 Tissue Fixation The technique of using FIXATIVES in the preparation of cytologic, histologic, or pathologic specimens for the purpose of maintaining the existing form and structure of all the constituent elements. Fixation, Tissue
D046529 Microscopy, Electron, Transmission Electron microscopy in which the ELECTRONS or their reaction products that pass down through the specimen are imaged below the plane of the specimen. Electron Diffraction Microscopy,Electron Microscopy, Transmission,Microscopy, Electron Diffraction,Transmission Electron Microscopy,Diffraction Microscopy, Electron,Microscopy, Transmission Electron

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