Structural and antigenic preservation of plant samples by microwave-enhanced fixation, using dedicated hardware, minimizing heat-related effects.
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.
Lería
Francisca
F
Centro de Investigaciones Biológicas (CSIC), E-28040 Madrid, Spain.
Marco
Roberto
R
Medina
Francisco Javier
FJ
D000490:Allium / Q000737:chemistry / Q000648:ultrastructure*; D000941:Antigens / Q000032:analysis*; D005404:Fixatives; D005557:Formaldehyde / Q000737:chemistry; D046529:Microscopy, Electron, Transmission; D008872:Microwaves; D011108:Polymers / Q000737:chemistry; D016707:Tissue Fixation / Q000379:methods*
D000941:Antigens; D005404:Fixatives; D011108:Polymers; D005557:Formaldehyde; C003043:paraform