A comparison of heating modes in rapid fixation techniques for electron microscopy.

Leonard, Jonathan B.; Shepardson, Sally

doi:10.1177/42.3.8308256

Cited by 19 publications

(7 citation statements)

References 11 publications

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“…This is contrary to conventional heating, which begins outside the sample (Leonard and Shepardson, 1994; Giberson and Demaree, 1995; Kok and Boon, 2003; De La Hoz et al, 2005; Schroeder et al, 2006; Zechmann and Zellnig, 2009). Due to this phenomenon, tissue processed in the microwave promotes deeper penetration of reagents.…”

Section: Discussionmentioning

confidence: 91%

Microwave processing of gustatory tissues for immunohistochemistry

Bond

Kinnamon

2013

Journal of Neuroscience Methods

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We use immunohistochemistry to study taste cell structure and function as a means to elucidate how taste receptor cells communicate with nerve fibers and adjacent taste cells. This conventional method, however, is time consuming. In the present study we used taste buds from rat circumvallate papillae to compare conventional immunohistochemical tissue processing with microwave processing for the colocalization of several biochemical pathway markers (PLCβ2, syntaxin-1, IP3R3, α-gustducin) and the nuclear stain, Sytox. The results of our study indicate that in microwave versus conventional immunocytochemistry: (1) fixation quality is improved; (2) the amount of time necessary for processing tissue is decreased; (3) antigen retrieval is no longer needed; (4) image quality is superior. In sum, microwave tissue processing of gustatory tissues is faster and superior to conventional immunohistochemical tissue processing for many applications.

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Section: Discussionmentioning

confidence: 91%

Microwave processing of gustatory tissues for immunohistochemistry

Bond

Kinnamon

2013

Journal of Neuroscience Methods

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“…Curiously, previous experiments of microwave-enhanced fixation paid little attention to the reduction of the fixative concentration, setting it at similar levels as those used in conventional procedures (Leonard and Shepardson, 1994;Login and Dvorak, 1988;Login et al, 1990;Ohtani, 1991;Wild, 1991). It is true that proposals were made some years ago in favor of the ability of microwave irradiation to act as the only fixation agent, due to its effects on protein coagulation.…”

Section: Discussionmentioning

confidence: 94%

“…While the reason why microwave irradiation of a polar substance results in energy absorption and heating is evident and the physical mechanisms that are responsible for heat production are well understood (Boon and Kok, 1988), it is not clear whether heating is the only cause of the enhancing effect of microwaves on fixation or if some additional effects are involved and, in this case, what might be their nature. In fact, this remains the main open discussion on this topic (Feirabend et al, 1992;Kok and Boon, 1990b;Leonard and Shepardson, 1994;Medina et al, 1995;Wild, 1991). Otherwise, an excess of heat applied to the sample by microwave irradiation is commonly considered unacceptable, since it results in protein denaturation; in general, published procedures of microwave-enhanced fixation recommend keeping the temperature below 50°C (see also Moriguchi et al, 2002).…”

Section: Introductionmentioning

confidence: 95%

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

Lería

Marco

Medina

2004

Microscopy Res & Technique

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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.

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“…Conflicting ideas about the nature of microwave fixation remain on the mechanisms of the heating versus the microwave effects. The heating effect is believed to improve penetration of reagents and speed up fixation 44 . Other publications have suggested that the microwave effects were non-thermal based on a study of globular proteins 45 and an increased rate of bone demineralization independent of temperature 46 .…”

Section: Experimental Designmentioning

confidence: 99%

High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

et al. 2012

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Conventional heavy metal post staining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by Field Emission Scanning Electron Microscope (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscope (TEM) samples, our technique utilizes osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains including uranyl acetate, lead aspartate, copper sulfate and lead citrate produced clean, highly contrasted TEM and SEM samples of insect, fish, and mammalian nervous system. This protocol takes 7–15 days to prepare resin embedded tissue, cut sections and produce serial section images.

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A comparison of heating modes in rapid fixation techniques for electron microscopy.

Cited by 19 publications

References 11 publications

Microwave processing of gustatory tissues for immunohistochemistry

Microwave processing of gustatory tissues for immunohistochemistry

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

High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

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