New application of air-drying techniques for studying Ephemeroptera and Plecoptera eggs by scanning electron microscopy

Ubero-Pascal, Nicolás; Fortuño, José-Manuel; Puig, Mariàngels

doi:10.1002/jemt.20248

Cited by 22 publications

(24 citation statements)

References 18 publications

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“…The use of HMDS is also a safer and cost-effective alternative to CPD. Several previous studies have shown that the use of HMDS yields comparable results as those obtained with identical biological specimens or sample tissues prepared with CPD (Bray et al, 1993, Hochberg and Litvaitis, 2000, Kennedy et al, 1989, Nation, 1983, Slizova et al, 2003, Ubero-Pascal et al, 2005. The data presented here demonstrate that application of HMDS to LVSEM sample preparation is appropriate for preserving and drying mammalian eggs and early zygotes.…”

supporting

confidence: 73%

Ultrastructural analysis of egg membrane abnormalities in post-ovulatory aged eggs

Dalo¹,

McCaffery²,

Evans³

2008

Int. J. Dev. Biol.

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Microscopic analyses of mammalian gamete ultrastructure have provided among the most seminal insights into fertilization. Here we present a summary of ultrastructural studies of mammalian fertilization, together with a review of the effects of post-ovulatory aging in eggs, and our own results using scanning electron microscopy to examine the effects of post-ovulatory aging on the egg membrane topography. Our previous work detected two abnormalities in egg membrane function in aged eggs: aged eggs appeared to be less able to support sperm-egg membrane interaction, thus rendering the eggs less fertilizable, and aged eggs appear to have a reduced ability to prevent polyspermy at the level of the egg membrane, i.e., to establish a membrane block to polyspermy. In the work presented here, we tested the suitability of both environmental (ESEM) and low-vacuum (LVSEM) modes of the FEI Quanta 200 Environmental Scanning Electron Microscope to study mammalian gametes. While ESEM mode was not sufficient under the conditions we used to observe fine cell surface details, the LVSEM mode proved to be an excellent way to view egg membrane topography. Unfertilized aged eggs have an abnormally distended amicrovillar region over the meiotic spindle, and fertilized aged eggs had three different abnormalities detected, from reduced sperm-induced membrane remodeling to abnormal sperm-induced membrane remodeling and membrane blebbing. Combining these insights of egg membrane function and topography with others in the field of post-ovulatory aging, we have expanded insights into why fertilization at increased times after ovulation is associated with poor reproductive outcomes.

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supporting

confidence: 73%

Ultrastructural analysis of egg membrane abnormalities in post-ovulatory aged eggs

Dalo¹,

McCaffery²,

Evans³

2008

Int. J. Dev. Biol.

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“…They were selected, rinsed, euthanised in water near to boil (except pupae), and fixed in McDowell fixative solution (4% formalin, 1% glutaraldehyde mixture in cacodylate buffer solution, pH 7.4) [46], at 4 8C for 24 h. Specimens were rinsed again twice in sodium cacodylate + sucrose solution 0.1 M, dehydrated until absolute acetone in a gradient of increasing concentration of ethanol and ethanol-acetone solution (50% of each reagent) and dried using critical point method or by air-drying after hexamethyldisilizane treatment [47]. Dried specimens were mounted on SEM stubs with conductive adhesive tape, mounting them in different positions (dorsal, lateral and ventral views).…”

Section: Methodsmentioning

confidence: 99%

Morphology of preimaginal stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae): A comparative study

Ubero-Pascal¹,

López-Esclapez²,

García³

et al. 2012

Forensic Science International

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“…In order to reduce costs and provide a similar effect to that proposed in Fang et al (2002), tetramethylsilane (TMS) will be used as a strong dehydrating agent that maintains tissue structure (Ubero-Pascal et al, 2005). Moreover, a series of investigations is set up to determine the specific way in which formalin acts to prevent amplification.…”

Section: Introductionmentioning

confidence: 99%

DNA extraction from formalin-fixed tissue: new light from the deep sea

Palero¹,

Hall²,

Clark³

et al. 2010

Sci. Mar.

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SUMMARY: DNA samples were extracted from ethanol and formalin-fixed decapod crustacean tissue using a new method based on Tetramethylsilane (TMS)-Chelex. It is shown that neither an indigestible matrix of cross-linked protein nor soluble PCR inhibitors impede PCR success when dealing with formalin-fixed material. Instead, amplification success from formalin-fixed tissue appears to depend on the presence of unmodified DNA in the extracted sample. A staining method that facilitates the targeting of samples with a high content of unmodified DNA is provided.Keywords: tetramethylsilane, ethidium bromide, formalin, Carcinus, Lithodidae. RESUMEN: Extracción de ADN a partir de tejido fijado en formol: nueva luz desde el mar abisal. -Muestras de ADN de distintos crustáceos decápodos fueron obtenidas independientemente a partir de tejidos fijados en etanol y tejidos fijados en formol mediante un nuevo protocolo basado en el Tetrametilsilano (TMS)-Chelex. Los resultados obtenidos muestran que el ADN no se encuentra atrapado de forma irreversible en una matriz proteica y que el éxito de amplificación no depende de la extracción de inhibidores de PCR solubles. Sin embargo, nuestros resultados indican que el éxito de amplificación depende de la presencia de ADN no modificado en la muestra. Se incluye un sencillo método de tinción que facilita la identificación de muestras con un alto contenido en ADN no modificado.Palabras clave: tetrametilsilano, bromuro de etidio, formol, Carcinus, Lithodidae.

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New application of air-drying techniques for studying Ephemeroptera and Plecoptera eggs by scanning electron microscopy

Cited by 22 publications

References 18 publications

Ultrastructural analysis of egg membrane abnormalities in post-ovulatory aged eggs

Ultrastructural analysis of egg membrane abnormalities in post-ovulatory aged eggs

Morphology of preimaginal stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae): A comparative study

DNA extraction from formalin-fixed tissue: new light from the deep sea

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