At the end of the protocol, the rats were sacrificed by lethal anaesthesia with an injection of 40mg/kg of pentobarbital solution (CEVA, Libourne, France). The heart was transversally divided into two parts: the base part was embedded into Tissue-Tek optimal cutting temperature (OCT) compound (VWR Chemicals, Jeuven, Belgium) and frozen into liquid nitrogen pre-cooled isopentane; the apex was snap-frozen in liquid nitrogen. All samples were stored at −80°C for further analyses. Heparin blood was collected from the left carotid artery at the end of the experiment, centrifuged at 3500 rpm for 15 min at 4°C, and plasma was stored at -80°C.
Tissue tek optimal cutting temperature compound
Manufactured by Avantor
Sourced in Belgium, United Kingdom, Germany
Tissue-Tek optimal cutting temperature (OCT) compound is a water-soluble, glycol-based medium used for the cryostat sectioning of tissue samples. It is designed to provide support and stabilization for the tissue during the freezing and sectioning process.
Automatically generated - may contain errors
5 protocols using tissue tek optimal cutting temperature compound
1
Cardiac Tissue Collection and Preservation
2
Spatial Transcriptomics of Ovarian Aging
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Ovaries were retrieved from Young and Aged females for spatial transcriptomic analysis for a total of 8 ovary sections from 8 mice (n = 4 per age group). Ovaries were rinsed in cold PBS, cryo-preserved in TissueTek Optimal Cutting Temperature (OCT) compound (VWR International), and stored at −80°C in an airtight container, as recommended by the manufacturer protocol (10x Genomics, Visium Spatial, CG000240 Rev C). Sections were selected for sequencing based on previous optimization of the sectioning depth required to reach the approximate midpoint of each ovary, based on the widest diameter region of the tissue.
3
Extensor Digitorum Longus Muscle Analysis in LAT1 mKO Mice
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LAT1 mKO mice were provided as a gift by Dr. Peter Taylor of the University of Dundee. Methods of generation of this mouse strain have been previously described [14 (link)]. Mice (n = 2 mKO, n = 2 wild type (WT)) were housed in a climate-controlled facility at the University of California Davis, in a standard 12 h light/dark cycle and fed standard chow ad libitum. All procedures were approved by the UC Davis Institutional Animal Care and Use Committee (IACUC) and performed under protocol number 19244. At three months of age, mice were fasted for 5 h before the extensor digitorum longus (EDL) muscle was surgically removed under anaesthetic (2.5% isofluorane), pinned to cork and frozen in liquid nitrogen-cooled isopentane. Muscles were subsequently stored at −80 °C until sectioned. Prior to sectioning, the belly of the EDL was blocked in Tissue-Tek Optimal Cutting Temperature (OCT) Compound (VWR International, Leicestershire, UK) and frozen in liquid nitrogen-cooled isopentane, at which point the sample was ready for cryo-sectioning. During staining protocols, both WT and mKO samples were processed and stained simultaneously, and image capture paradigms were also identical.
4
Nestin Expression in Mouse Brain
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Mice were anesthetized and cardiac perfused through the heart with 4% paraformaldehyde. Brains were dissected out, post-fixed and stored in 30% sucrose. Brains were frozen in Tissue-Tek optimal cutting temperature compound (O.C.T.) (VWR, Richmond, IL) and coronally sectioned at a 25 μm interval. Tissue sections were blocked with 3% normal donkey serum (NDS, Jackson) in PBS for 30 minutes and then incubated with Nestin (Abcam; 1:3000) at 4 °C overnight followed by CF488 (Biotium; 1:1000) for 1 hour. DAPI was used as counter staining. Images were acquired and analyzed on a Leica TCS SP2 confocal fluorescence microscope.
5
Transplantation of Pig Islets in Diabetic Mice
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Animal research was approved by the Regional Administrative Council Giessen, Veterinary Department under the code GI20/11Nr.15/2006 and performed in accordance with the German Animal Welfare law and the ARRIVE guidelines. Diabetes was induced in NRMI nu/nu mice at the age of 12 weeks by a single injection of 180 mg/kg streptozotocin (Sigma, MS, USA) intraperitoneally. Blood glucose levels were monitored using the glucometer Elite (Bayer, Leverkusen, Germany). Mice with a non-fasting blood glucose concentration of more than 16.7 mM for three consecutive days were selected for transplantation (n = 16). Mice were divided into two groups and treated i.p. with 0.12 mg/kg/day thioredoxin reductase inhibitor auranofin (0.2 mg/ml in 0,9% saline, dissolved in 0.25 ml ethanol and 0.2 ml Cremophor EL) or vehicle (n = 8 per group) throughout the study period. Recipients were anaesthetized with avertine and maintained with isoflurane. 2000 pancreatic pig islet equivalents were transplanted into the liver via the portal vein with a 27-gauge needle as previously described [29 ]. Grafted livers and portal vein blood were recovered 30 min after transplantation of pancreatic islets. Healthy pancreata were used as controls. Grafted livers were embedded in TissueTek optimal cutting temperature compound (VWR International GmbH, Darmstadt, Germany) and snap-frozen in liquid nitrogen.
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