At the end of the ICV infusion, the brains were removed and divided at the point of bregma after perfusion with phosphate‐buffered saline, and the caudal side of each brain was immediately frozen in Tissue‐Tek OCT embedding medium (Sakura Finetek, Tokyo, Japan). An 8‐μm slice was made at 1.43 to 2.43 mm caudally from the bregma for the following histological evaluations. In addition, the cardiac left ventricle and gastrocnemius muscle from each mouse were removed, weighed, and frozen in Tissue‐Tek OCT embedding medium (Sakura Finetek, Tokyo, Japan) and in paraffin for the following histological evaluations.
Tissue tek oct embedding medium
Manufactured by Sakura Finetek
Sourced in Japan
Tissue-Tek OCT embedding medium is a cryogenic embedding compound used to prepare tissue samples for frozen sectioning. It is designed to provide consistent, uniform support and embedding of a wide variety of tissues for optimal sectioning and preservation of tissue morphology.
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Lab products found in correlation
Straight spring wire, by Cook Medical (2 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Dihydroethidium dhe, by Beyotime (1 mentions)
Ab203591, by Abcam (1 mentions)
Von willebrand factor antibody, by Abcam (1 mentions)
8 ohdg, by Abcam (1 mentions)
Dcfh da ros assay kit, by Beyotime (1 mentions)
Cm3050 cryostat, by Leica (1 mentions)
Vectamount aq medium, by Vector Laboratories (1 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
Dihydroethidium dhe staining, by Merck Group (1 mentions)
Tcs sp5 confocal microscopy, by Leica (1 mentions)
Las af lite software, by Leica (1 mentions)
Glomax, by Promega (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Carl Roth (1 mentions)
Rompun 2, by Bayer (1 mentions)
15 protocols using tissue tek oct embedding medium
1
Brain Tissue Preparation for Histological Analysis
2
Histochemical Analysis of Liver Lipids
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Dissected liver sections were fixed overnight with 4% paraformaldehyde, cryoprotected with 15%-30% sucrose and embedded in Tissue-Tek OCT embedding medium (Sakura Finetek Europe, The Netherlands). Cryostat sections (8-10 microns) were rinsed with PBS and stained with a 0.3% solution of Oil Red O for 10 minutes at room temperature. After washing in PBS, the sections were counterstained with hematoxylin and eosin for 1 minute and washed with water. 31 (link) The percentage of the stained area was determined using Image J software.
3
Cellular Localization of CsNps in Mouse Liver
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To detect the cellular location of the CsNps in mouse liver, the mice (n=3) were intravenously injected with RBITC-CsNps at a dose of 0.16 g/kg. After 3 hrs, 12 hrs, 1 day, 3 days, 5 days and 30 days, the mice were sacrificed, and the liver was excised and immediately frozen in Tissue-Tek OCT embedding medium (Sakura Finetek, Torrance, CA, USA) for 8 μm cryo-sectioning. Kupffer cells in the sections were analyzed by immunofluorescence staining with mouse anti-mouse CD68 monoclonal antibody (Abcam, Shanghai, China) followed by rabbit anti-mouse kFlour647-labeled anti-IgG antibody (KeyGene Biotec, Nanjing, China). The hepatocytes were analyzed by immunofluorescence staining with mouse Anti-CK18 followed by goat anti-mouse IgG(H+L)/FITC (KeyGene Biotec). The nuclei in the sections were stained with DAPI, and the sections were visualized on a laser confocal microscope.
Considering the tissue sections in this test were only 8 μm, to increase the fluorescence signals in the very thin sections, the dosage was set as 0.16 g/kg, four times higher than the following animal tests, in which the signals were detected in the whole tissues or in feces/urines.
Considering the tissue sections in this test were only 8 μm, to increase the fluorescence signals in the very thin sections, the dosage was set as 0.16 g/kg, four times higher than the following animal tests, in which the signals were detected in the whole tissues or in feces/urines.
4
Aortic Superoxide Quantification in Rats
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Hearts or 4 mm vessel rings of thoracic aorta removed from rats were immediately frozen in Tissue-Tek OCT embedding medium (Sakura Finetek, Tokyo, Japan). Then, the samples were cut into 5-μm-thick sections and placed on glass slides. Dihydroethidium (DHE, 2 μM, Beyotime, China), topically applied to each tissue section, was used to evaluate superoxide levels in situ. After the slides were incubated in a dark chamber at 37°C for 30 min, DHE fluorescences were observed by fluorescence microscope (Olympus, Japan).
5
Quantification of KLF2 in Ischemic Rat MCA
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Segments of the ipsilateral MCA from SHR rats subjected to ischaemia (90 min)/reperfusion (30 min) were exposed ex vivo to either vehicle or UA (30 µM) added into the culture medium for 23.5 h. Afterwards, segments were fixed in 4% paraformaldehyde and embedded in Tissue Tek OCT embedding medium (Sakura Finetek Europe, Zoeterwoude, the Netherlands), frozen in liquid nitrogen and kept at -70 °C. Frozen transverse sections (14 μm-thick) were incubated for 1 h with anti-KLF2 (1:100; #Ab203591, lot GR313218-2, Abcam) primary antibody in a humidified chamber at 37 °C. After several rinses, sections were incubated (for 45 min) with a donkey anti-rabbit IgG secondary antibody conjugated to Cyanine 3 (1:200; #711-165-152, lot 110351, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in a humidified chamber at 37°C. Sections were processed for immunofluorescence staining as previously described [21] . The specificity of the immunostaining was verified by omission of the primary antibody. Images were captured using a FV1000 confocal microscope (Olympus Iberia, Barcelona, Spain). Quantitative analysis of average fluorescence signal was obtained in at least two rings of each animal with Image J software (National Institutes of Health, Bethesda, MD, USA), and the results were expressed as arbitrary units.
6
Immunofluorescent Analysis of Oxidative Stress
7
Quantifying Cellular Oxidative Stress
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Muscle tissues were frozen in Tissue-Tek OCT embedding medium (Sakura Finetek, Tokyo, Japan) and cut into 8μm thick sections. 10 μM dihydroethidium (DHE, Beyotime, China) was used to evaluate ROS levels in situ by incubating the slides in a dark chamber at 37 °C for 30 min 14 (link). 2 μM DHE and 10 μM DCFH-DA (ROS Assay Kit, Beyotime, China) were used to incubate the in vitro cells at 37 °C for 20 min for ROS analyses as manufacturing instructions.
8
Immunohistochemical Analysis of Liver Tissue
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Liver tissues were harvested and transferred to Tissue-Tek OCT embedding medium (Sakura Finetek, Torrance, CA), and snap-frozen in iso-pentane chilled in dry ice. Cryosections (4μm) were cut using a Leica CM 3050 cryostat (Leica Microsystems, Nussloch, Germany). The sections were air-dried and fixed with acetone for 10 min. Tissue sections were rehydrated with PBS and incubated with the primary antibody in appropriate dilution (refer to S1 Table
9
Superoxide Evaluation in Cardiac Tissue
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Hearts and aortas removed from DS rats were immediately frozen in Tissue-Tek OCT embedding medium (Sakura Finetek, Tokyo, Japan). Dihydroethidium (DHE) was used to evaluate tissue superoxide levels in situ, as described [32 (link)]. In brief, DHE fluorescence was visualized by fluorescence microscopy using an excitation wavelength of 520–540 nm and a rhodamine emission filter. DHE fluorescence of tissue was captured with the same exposure time (1.0 s), and it was quantified using Lumina Vision. The mean fluorescence was quantified and expressed relative to values obtained from control rats. We have previously verified that DHE fluorescence obtained by our method is indeed attributed to superoxide [33 (link)].
10
In Situ Superoxide Quantification in Brain Tissues
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At the end of the experiment, the brains were removed and divided at the point of bregma, and the caudal side of each brain was immediately frozen in Tissue‐Tek OCT embedding medium (Sakura Finetek, Tokyo, Japan). Dihydroethidium (DHE) was used to evaluate tissue superoxide levels in situ as described.24 DHE fluorescence of each tissue section in the 2 fields of the hippocampal CA1 region and somatosensory cortex on both sides for each animal at ×200 magnification was quantified by Win ROOF version 5.8 analysis software (Mitani Corporation, Tokyo, Japan). Mean fluorescence was quantified and expressed relative to values obtained from wild‐type mice fed control diet. To confirm that DHE fluorescence was derived from superoxide, brain sections were preincubated with 250 U/mL polyethylene‐glycol superoxide dismutase (SOD) (Sigma‐Aldrich, Saint Louis, MO, USA) for 30 minutes followed by DHE staining, according to our previous method.25 , 26 , 27
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