Novel zinc-based fixative for high quality DNA, RNA and protein analysis

Lykidis, Dimitrios; Noorden, Susan Van; Armstrong, Alan; Spencer‐Dene, Bradley; Li, Jie; Zhuang, Zhengping; Stamp, Gordon

doi:10.1093/nar/gkm433

Cited by 85 publications

(87 citation statements)

References 11 publications

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“…For immunohistochemical analyses, ethanol-and ZBFfixed tissue is found to be advantageous, often eliminating the need for antigen-retrieving procedures commonly performed on NBF-fixed tissue (Beckstead 1994;Ismail et al 2003;Hicks et al 2006). It has also been found that nucleic acids are better preserved in ethanol-and ZBF-fixed material than in NBF-fixed material (Gillespie et al 2002;Leiva et al 2003;Wester et al 2003;Lykidis et al 2007). In the present study, we have shown that paraffin-embedded, ethanol-or ZBF-fixed tissue is also well suited for in situ zymography.…”

Section: Discussionmentioning

confidence: 99%

Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

Hadler‐Olsen

Kanapathippillai

Berg

et al. 2009

J Histochem Cytochem.

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In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue.

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Section: Discussionmentioning

confidence: 99%

Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

Hadler‐Olsen

Kanapathippillai

Berg

et al. 2009

J Histochem Cytochem.

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“…Cells were resuspended in permeabilization buffer (PB, PBS supplemented with 0.1% BSA and 0.2% saponin) for 10 min on ice. After centrifugation, cells were stored at −20°C in a modified zinc-based fixative (0.5% zinc chloride, 0.5% zinc trifluoroacetate, 0.05% calcium acetate in 0.1M Tris-HCL, pH to 6.4–6.7) and glycerol (1:1), as previously described (Jensen et al 2010; Lykidis et al 2007), until further analysis.…”

Section: Methodsmentioning

confidence: 99%

Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week

Nikodémová

Kimyon

et al. 2015

Journal of Neuroimmunology

145

109

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During postnatal development, microglia, CNS resident innate immune cells, are essential for synaptic pruning, neuronal apoptosis and remodeling. During this period microglia undergo morphological and phenotypic transformations; however, little is known about how microglial number and density is regulated during postnatal CNS development. We found that after an initial increase during the first 14 postnatal days, microglial numbers in mouse brain began declining in the third postnatal week and were reduced by 50% by 6 weeks of age; these “adult” levels were maintained until at least 9 months of age. Microglial CD11b levels increased, whereas CD45 and ER-MP58 declined between P10 and adulthood, consistent with a maturing microglial phenotype. Our data indicate that both increased microglial apoptosis and a decreased proliferative capacity contribute to the developmental reduction in microglial numbers. We found no correlation between developmental reductions in microglial numbers and brain mRNA levels of Cd200, Cx3Cl1, M-Csf or Il-34. We tested the ability of M-Csf-overexpression, a key growth factor promoting microglial proliferation and survival, to prevent microglial loss in the third postnatal week. Mice overexpressing M-Csf in astrocytes had higher numbers of microglia at all ages tested. However, the developmental decline in microglial numbers still occurred, suggesting that chronically elevated M-CSF is unable to overcome the developmental decrease in microglial numbers. Whereas the identity of the factor(s) regulating microglial number and density during development remains to be determined, it is likely that microglia respond to a “maturation” signal since the reduction in microglial numbers coincides with CNS maturation.

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“…diameter 1 cm) were immersed in either liquid nitrogen (n=16), zinc-based Z7 fixative (n=11),3 RCL2 (n=11; Alphelys, Plaisir, France), PAXgene (n=10; QIAGEN, Hilden, Germany), Allprotect (n=5; QIAGEN) and RNAlater (n=5; Invitrogen, Life Technologies, UK) or in 10% neutral buffered formaldehyde (NBF; n=11). The Z7 fixative was used as previously described3 and other fixatives (RCL2, PAXgene, Allprotect and RNAlater) were used according to manufacturer's instructions. All samples were dehydrated and paraffin-embedded (Shandon Histoplast paraffin, Thermo Scientific) after fixation.…”

Section: Methodsmentioning

confidence: 99%

Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives

et al. 2013

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Formalin fixation preserves tissue morphology at the expense of macromolecule integrity. Freshly frozen samples are the golden standard for DNA and RNA analyses but require laborious deep-freezing and frozen sectioning for morphological studies. Alternative tissue stabilisation methods are therefore needed. We analysed the preservation of nucleic acids, immunohistochemical staining properties and tissue morphology in paraffin-embedded clinical tissue samples fixed with Z7, RCL2, PAXgene, Allprotect and RNAlater. Formalin-fixed and deep-frozen samples were used as controls. Immunohistochemical analyses showed good preservation of antigenicity in all except Allprotect and RNAlater-fixed samples. RNA quality, based on RNA integrity number value by Bioanalyzer, was comparable with freshly frozen samples only in PAXgene-fixed samples. According to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, RNA from PAXgene samples yielded results similar to freshly frozen samples. No difference between fixatives was seen in DNA analyses (PCR and real-time PCR). In conclusion, PAXgene seems to be superior to other molecular fixatives and formaldehyde.

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Novel zinc-based fixative for high quality DNA, RNA and protein analysis

Cited by 85 publications

References 11 publications

Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week

Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives

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