Comparative LC–MS/MS lipidomic analysis of macaque heart tissue flash frozen or embedded in optimal cutting temperature polymer (OCT): Practical considerations

2021 ◽  
Author(s):  
Ashish Vaswani ◽  
Armando Alcazar Magana ◽  
Eric Zimmermann ◽  
Wohaib Hasan ◽  
Jaishankar Raman ◽  
...  
Keyword(s):  
Cutting Temperature ◽  
Heart Tissue ◽  
Lipidomic Analysis ◽  
Optimal Cutting Temperature

scholarly journals Cryo-Gel embedding compound for renal biopsy biobanking

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Malou L. H. Snijders ◽  
Marina Zajec ◽  
Laurens A. J. Walter ◽  
Remco M. A. A. de Louw ◽  
Monique H. A. Oomen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cutting Temperature ◽  
Renal Tissue ◽  
Tissue Samples ◽  
Normal Spleen ◽  
Renal Biopsies ◽  
Optimal Cutting Temperature ◽  
Embedding Medium ◽  
Rna And Dna

Abstract Optimal preservation and biobanking of renal tissue is vital for good diagnostics and subsequent research. Optimal cutting temperature (OCT) compound is a commonly used embedding medium for freezing tissue samples. However, due to interfering polymers in OCT, analysis as mass spectrometry (MS) is difficult. We investigated if the replacement of OCT with Cryo-Gel as embedding compound for renal biopsies would enable proteomics and not disturb other common techniques used in tissue diagnostics and research. For the present study, fresh renal samples were snap-frozen using Cryo-Gel, OCT and without embedding compound and evaluated using different techniques. In addition, tissue samples from normal spleen, skin, liver and colon were analyzed. Cryo-Gel embedded tissues showed good morphological preservation and no interference in immunohistochemical or immunofluorescent investigations. The quality of extracted RNA and DNA was good. The number of proteins identified using MS was similar between Cryo-Gel embedded samples, samples without embedding compound and OCT embedded samples. However, polymers in the OCT disturbed the signal in the MS, while this was not observed in the Cryo-Gel embedded samples. We conclude that embedding of renal biopsies in Cryo-Gel is an excellent and preferable alternative for OCT compound for both diagnostic and research purposes, especially in those cases where proteomic analysis might be necessary.

scholarly journals A simple method for sphingolipid analysis of tissues embedded in optimal cutting temperature compound

2020 ◽  
Vol 61 (6) ◽  
pp. 953-967
Author(s):  
Timothy D. Rohrbach ◽  
April E. Boyd ◽  
Pamela J. Grizzard ◽  
Sarah Spiegel ◽  
Jeremy Allegood ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cutting Temperature ◽  
Protein Quantification ◽  
Sphingolipid Metabolism ◽  
Normal Lung ◽  
Human Tissues ◽  
Simple Method ◽  
Disease Etiology ◽  
Esi Ms ◽  
Optimal Cutting Temperature

MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of OCT compound used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT compound also interferes with protein quantification assays, and that the presence of OCT compound impacts the quantification of extracted sphingolipids by LC-ESI-MS/MS. We developed and validated a simple and inexpensive method that removes OCT compound from OCT compound-embedded tissues. Our results indicate that removal of OCT compound from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC-ESI-MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer and to determine the long-term stability of sphingolipids in OCT compound-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT compound-cryopreserved normal lung tissues. This validated sphingolipidomic OCT compound-removal protocol should be a valuable addition to the lipid biologist’s toolbox.

scholarly journals Quantitative Proteomic Analysis of Optimal Cutting Temperature (OCT) Embedded Core-Needle Biopsy of Lung Cancer

2017 ◽  
Vol 28 (10) ◽  
pp. 2078-2089 ◽  
Author(s):  
Xiaozheng Zhao ◽  
Kenneth E. Huffman ◽  
Junya Fujimoto ◽  
Jamie Rodriguez Canales ◽  
Luc Girard ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Core Needle Biopsy ◽  
Proteomic Analysis ◽  
Needle Biopsy ◽  
Cutting Temperature ◽  
Core Needle ◽  
Quantitative Proteomic Analysis ◽  
Optimal Cutting Temperature

scholarly journals Fast Localization and Sectioning of Mouse Locus Coeruleus

2020 ◽  
Vol 2020 ◽  
pp. 1-5
Author(s):  
Song Cao ◽  
Juan Li ◽  
Jie Yuan ◽  
Dexin Zhang ◽  
Tian Yu
Keyword(s):  
Fourth Ventricle ◽  
Brain Slices ◽  
Cutting Temperature ◽  
Cutting Plane ◽  
Noradrenergic Neurons ◽  
Noradrenergic Neuron ◽  
Easy Method ◽  
Adult Mice ◽  
Optimal Cutting Temperature ◽  
The Brain

The locus nucleus (LC) is a multifunctional nucleus which is also the source of norepinephrine in the brain. To date, there is no simple and easy method to locate the small LC in brain sectioning. Here we report a fast, accurate, and easy-to-follow protocol for the localization of mice LC in frozen sectioning. After fixation and dehydration, the intact brains of adult mice were placed on a horizontal surface and vertically cut along the posterior margin of the bilateral cerebral cortex. In the coronal cutting plane, the aqueduct of midbrain can be seen easily with the naked eyes. After embedding the cerebellum part with optimal cutting temperature (OCT) compound, coronal brain slices were cut from the cutting plane, within 1 mm, the aqueduct of midbrain disappeared and the fourth ventricle appeared, then the brain slices contained LC and were collected. From the first collection, at ~200 μm, the noradrenergic neurons’ most enriched brain slices can be collected. The tyrosine hydroxylase immunofluorescence staining confirmed that the localization of LC with this method is accurate and the noradrenergic neuron most abundant slices can be determined with this method.

scholarly journals Authentication of In Situ Measurements for Thoracic Aortic Aneurysms in Mice

2020 ◽  
Author(s):  
Satoko Ohno-Urabe ◽  
Masayoshi Kukida ◽  
Michael K. Franklin ◽  
Hong S. Lu ◽  
Alan Daugherty ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Left Ventricle ◽  
Cutting Temperature ◽  
Endothelial Damage ◽  
Aortic Aneurysms ◽  
Thoracic Aortic Aneurysms ◽  
Ultrasound Measurements ◽  
Standard Parameter ◽  
Optimal Cutting Temperature

AbstractAortic diameter is a standard parameter for defining disease severity of thoracic aortic aneurysms. In mouse studies, aortic diameters can be measured directly in situ, but this approach has the potential confounder of underestimation due to the absence of physiological arterial pressure. In the present study, we developed an in situ approach for authentic aortic measurements. Thoracic aortic aneurysms were induced by β-aminopropionitrile (BAPN, 0.5% wt/vol) administration in 4-week-old male C57BL/6J mice. Ultrasonography was performed to examine aortic dimensions, and mice with thoracic aortic dilatations were terminated subsequently. After saline perfusion through the left ventricle, periaortic tissues were removed to expose thoracic aortas. Optimal cutting temperature (OCT) compound was injected via the left ventricle to maintain aortic patency. In situ aortic images were captured pre- and post-OCT injection. In mice with severe thoracic aortic aneurysms, smaller aortic diameters were observed prior to OCT injection compared to ultrasound measurements, while aortic diameters in situ after OCT were comparable to diameters measured using ultrasound. Immunostaining for CD31 revealed that endothelial cells were preserved in the intima after OCT injection, indicating that OCT injection does not cause endothelial damage. In conclusion, in situ imaging with OCT injection provides authentic aortic measurements without overt aortic damage in mice with thoracic aortic aneurysms.

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