Retrospective electron microscopy: Preservation of fine structure by freezing and aldehyde fixation

Fortunato, Franco; Hackert, Thilo; Büchler, Markus W.; Kroemer, Guido

doi:10.1080/23723556.2016.1251382

Cited by 9 publications

(6 citation statements)

References 17 publications

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“…2 , 3 , 4 , 5 ) or frozen samples processed with AFS (Supplemental Figures S1 – S3). In contrast to conventional fixation or another published method fixating at 4 °C (Fortunato et al 2016 ), our preparation method for cryo-stored samples uses a higher temperature (37 °C instead of room temperature as normally used in non-frozen samples) during chemical fixation, with the rationale to facilitate an accelerated thawing process. Both, frozen as well as fresh samples which underwent chemical fixation were fixated in 2.5% glutaraldehyde/2% paraformaldehyde (Fig.…”

Section: Resultsmentioning

confidence: 99%

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy

Galhuber

Kupper

Dohr

et al. 2021

Histochem Cell Biol

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Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. “biobanking” of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.

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Section: Resultsmentioning

confidence: 99%

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy

Galhuber

Kupper

Dohr

et al. 2021

Histochem Cell Biol

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“…U2OS cells (2 × 10 6 ) were seeded in petri dishes (Greiner Bio‐One) and let to adapt for 24 h. Redaporfin (5 μM) was added and photoactivation was performed as mentioned above. Six hours later, cells were fixed in 1.6% glutaraldehyde (v/v in 0.1 M phosphate buffer) for 1 h, collected by scraping from the petri dish, and centrifuged, and the pellet was post‐fixed with 1% osmium tetroxide (w/v in 0.1 M phosphate buffer) for 2 h. Following dehydration through a graded ethanol series, cells were embedded in Epon™ 812 and ultrathin sections were stained with standard uranyl acetate and lead citrate (Fortunato et al , ). Images were acquired using a Tecnai 12 electron microscope (FEI, Eindhoven, the Netherlands).…”

Section: Methodsmentioning

confidence: 99%

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

et al. 2018

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Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.

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“…Transmission electron microscopy (TEM). Frozen liver samples were prepared and fixed in 1.5% paraformaldehyde and incubated at 4°C overnight 93 , after which fixed tissues were processed usinf a protocol based on ref. 94 .…”

Section: Network Analysesmentioning

confidence: 99%

Transkingdom interactions between Lactobacilli and hepatic mitochondria attenuate western diet-induced diabetes

et al. 2021

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Western diet (WD) is one of the major culprits of metabolic disease including type 2 diabetes (T2D) with gut microbiota playing an important role in modulating effects of the diet. Herein, we use a data-driven approach (Transkingdom Network analysis) to model host-microbiome interactions under WD to infer which members of microbiota contribute to the altered host metabolism. Interrogation of this network pointed to taxa with potential beneficial or harmful effects on host’s metabolism. We then validate the functional role of the predicted bacteria in regulating metabolism and show that they act via different host pathways. Our gene expression and electron microscopy studies show that two species from Lactobacillus genus act upon mitochondria in the liver leading to the improvement of lipid metabolism. Metabolomics analyses revealed that reduced glutathione may mediate these effects. Our study identifies potential probiotic strains for T2D and provides important insights into mechanisms of their action.

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Retrospective electron microscopy: Preservation of fine structure by freezing and aldehyde fixation

Cited by 9 publications

References 17 publications

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

Transkingdom interactions between Lactobacilli and hepatic mitochondria attenuate western diet-induced diabetes

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