Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Background: Uric acid (UA) concentration within carotid plaque and its association with cerebrovascular events have not been detected or quantified. Systemically, serum UA is a marker of inflammation and risk factor for atherosclerosis. However, its association with carotid plaque instability and stroke pathogenesis remains unclear. In patients undergoing carotid endarterectomy, we aimed to determine whether UA is present differentially in symptomatic versus asymptomatic carotid plaques and whether serum UA is associated with cerebrovascular symptoms (stroke, transient ischemic attack, or amaurosis fugax). Methods: Carotid atherosclerotic plaques were collected during carotid endarterectomy. The presence of UA was assessed using Gomori methenamine silver staining as well as anti-UA immunohistochemical staining and its quantity measured using an enzymatic colorimetric assay. Clinical information was obtained through a retrospective review of data. Results: UA was more commonly detected in symptomatic (n=23) compared with asymptomatic (n=9) carotid plaques by Gomori methenamine silver (20 [86.9%] versus 2 [22.2%]; P =0.001) and anti-UA immunohistochemistry (16 [69.5%] versus 1 [11.1%]; P =0.004). UA concentration was higher in symptomatic rather than asymptomatic plaques (25.1 [9.5] versus 17.9 [3.8] µg/g; P =0.021). Before carotid endarterectomy, serum UA levels were higher in symptomatic (n=341) compared with asymptomatic (n=146) patients (5.9 [interquartile range, 4.6–6.9] mg/dL versus 5.2 [interquartile range, 4.6–6.2] mg/dL; P =0.009). Conclusions: The current study supports a potential role of UA as a potential tissue participant and a systemic biomarker in the pathogenesis of carotid atherosclerosis. UA may provide a mechanistic explanation for plaque instability and subsequent ischemic cerebrovascular events.

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“…Extended methods can be found in the Supplemental Material, Supplemental Methods with the following references. 16,35–52…”

Section: Methodsmentioning

confidence: 99%

Uric Acid Expression in Carotid Atherosclerotic Plaque and Serum Uric Acid Are Associated With Cerebrovascular Events

et al. 2022

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“…For extracting genomic DNA, 5–10 sections from each block were taken and put into sterile, nuclease-free Eppendorf tubes. Prior to genomic DNA extraction, xylene and paraffin residues from paraffin sections were removed in the pathology lab in accordance with standard operating procedures [26]. To quickly and effectively deparaffinize the tissue, 1 mL of xylene was added to each tube, and the tubes were vortexed violently for 10 seconds.…”

Section: Methodsmentioning

confidence: 99%

A Genetic and Immunohistochemical Analysis ofH. PyloriPhenotypes and p27 expression in Adenocarcinoma Patients in Jordan

Brown

Mohammad

Shboul

et al. 2022

Preprint

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Gastric cancer is one of the most prevalent malignant cancers worldwide and specifically, adenocarcinoma. Based on prior research, there is an association between Helicobacter pylori (H. pylori) infection and the frequency of duodenal ulcer, distal gastric adenocarcinoma, mucosa-associated lymphoid tissue lymphoma, and antral gastritis. H. pylori virulence and toxicity factors have been identified that significantly influence the clinical outcomes of H. pylori infection and gastric adenocarcinoma. However, it is unclear exactly how different strains of H. pylori infection affect gastric adenocarcinoma. Current research suggests this involves tumor suppressor genes, like p27, but also H. Pylori toxic proteins. Therefore, we quantified known H. Pylori genotypes within adenocarcinoma patients to establish the prevalence of known toxins that include cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) within patients of variable diagnosis. This analysis used gastrectomy samples which were validated for DNA viability. The incidence of H. Pylori in adenocarcinoma patients in Jordan was established to be 54.5% positive (ureA gene) with cagA genotype occurrence at 57.1% but also vacA gene ratios 24.7%:22.1%: 14.3%:14.3%. (vacAs1:vacAs2: vacAm1:vacAm2). It is statistically significant that p27 was dysregulated and suppressed within nearly all H. Pylori vacA genotypes but also that according to our analysis that 24.6% of H. Pylori samples analyzed had an unknown novel genotype and curiously that p27 protein expression was retained in only 12% of tested adenocarcinoma H. Pylori samples which is suggestive that p27 could be used as a prognostic indicator but also that an un-known yet genotype as yet not characterized could be contributing to the regulation of p27 protein within this cellular environment.

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“…Fixed mouse and human liver samples were processed using the standard paraffin embedding method 38 , cut into 10 μm sections using a microtome (Leica Biosystems, Germany) and placed on microscope slides (Fisher Scientific, CA, USA). Tissue sections were deparaffinized with standard protocol 39 , followed by heat-mediated epitope recovery (30 min, 100 °C) in anhydrous citric acid solution (1.92 g of anhydrous citric acid per 1000 ml of distilled water, pH 2). Liver sections were stained with hematoxylin and eosin (H&E) according to standardized procedure 40 .…”

Section: Histological Analysis Of Mouse and Human Liversmentioning

confidence: 99%

Diosmetin alleviates liver inflammation by improving liver sinusoidal endothelial cell dysfunction

Żurawek,

Pydyn,

Major

et al. 2023

Preprint

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Background & Aims: Tumor necrosis factor-alpha (TNFalpha) induces pro-inflammatory activation in liver sinusoidal endothelial cells (LSEC) and liver inflammation. However, knowledge about whether modulating LSEC activation can alleviate liver inflammation is scarce. This study aimed to establish and validate an animal model mimicking LSEC dysfunction observed in patients with elevated plasma levels of TNFalpha, and explore whether vasoactive flavonoid diosmetin could serve as a therapeutic agent for liver inflammation. Approach & Results: Genetic deletion of Mcpip1 in myeloid leukocytes (Mcpip1fl/flLysMCre) resulted in the development of systemic and liver inflammation in mice. Symptoms were compared with those in liver samples from obese humans with elevated TNFalpha. Mice were treated with diosmetin, and its effectiveness in alleviating liver inflammation was evaluated. Elevated TNFalpha correlated with reduced Mcpip1 expression in peripheral blood mononuclear cells and LSEC dysfunction in obese patients. Mcpip1 knockout in myeloid cells in mice replicated molecular signs observed in human samples. Diosmetin efficiently reduced LSEC activation and liver inflammation in Mcpip1fl/flLysMCre mice. Diosmetin's effects may stem from inhibiting NF-kappaB-p50 subunit production in TNFalpha-activated endothelial cells. Conclusions: Diosmetin treatment efficiently restricted liver inflammation, despite ongoing systemic inflammation, by diminishing LSEC dysfunction. Mcpip1fl/flLysMCre mice mimic symptoms of liver inflammation observed in humans and can be useful in studies on new anti-inflammatory therapies for the liver. We show that diosmetin, a vasoactive flavonoid that is successfully used in the clinic to treat chronic venous insufficiency, has also strong anti-inflammatory properties in the liver. This suggests that diosmetin treatment may be tested in humans as a supportive therapy for liver inflammation.

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Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Cited by 7 publications

References 7 publications

Uric Acid Expression in Carotid Atherosclerotic Plaque and Serum Uric Acid Are Associated With Cerebrovascular Events

Uric Acid Expression in Carotid Atherosclerotic Plaque and Serum Uric Acid Are Associated With Cerebrovascular Events

A Genetic and Immunohistochemical Analysis ofH. PyloriPhenotypes and p27 expression in Adenocarcinoma Patients in Jordan

Diosmetin alleviates liver inflammation by improving liver sinusoidal endothelial cell dysfunction

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