All invasive procedures were performed 48–72 h after a training session, between 7 and 10 a.m., and under standardized conditions after an overnight fast. A blood sample was collected from an antecubital vein, and a biopsy was collected at rest after 0, 16, and 52 wks from m. vastus lateralis under sterile conditions and local anesthesia (1% Lidocaine, Amgros 742122, Copenhagen, Denmark) using the Bergstrom technique [16 ]. A part of the muscle sample (40 mg wet weight) was immediately frozen in liquid N2 and stored at –80°C. The remainder of the muscle tissue was mounted in an embedding medium (OCT Tissue-Tek, Sakura Finetek, Zoeterwoude, NL) and frozen in pre-cooled isopentane and subsequently stored at –80°C until further analysis.
Tissue tek oct
Manufactured by Sakura Finetek
Sourced in United States, Japan, Netherlands, Germany
The Tissue-Tek OCT is a cryostat system designed for the preparation of frozen tissue sections. It provides a controlled environment for the freezing and sectioning of tissue samples, enabling their subsequent analysis and examination.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost plus slide, by Thermo Fisher Scientific (12 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (9 mentions)
Triton x 100, by Merck Group (6 mentions)
Superfrost plus adhesive slides, by Thermo Fisher Scientific (6 mentions)
Lsm 700 confocal microscope, by Zeiss (6 mentions)
Oil red o, by Merck Group (5 mentions)
Vectashield, by Vector Laboratories (5 mentions)
Fluoromount g, by Southern Biotech (5 mentions)
Positively charged slides, by Thermo Fisher Scientific (5 mentions)
Cryostat, by Leica camera (5 mentions)
Cy3 streptavidin, by Jackson ImmunoResearch (5 mentions)
Paraformaldehyde (pfa), by Merck Group (5 mentions)
Leica cm 1510s cryostat, by Leica Biosystems (4 mentions)
Cm3050s cryostat, by Leica camera (4 mentions)
Vectashield mounting medium, by Vector Laboratories (4 mentions)
Superfrost plus, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Carl axioplan2 imaging microscope b000707, by Zeiss (3 mentions)
Cm1900, by Leica Microsystems (3 mentions)
Peanut agglutinin, by Vector Laboratories (3 mentions)
333 protocols using tissue tek oct
1
Standardized Skeletal Muscle Biopsy
2
Quantifying In Situ ROS Synthesis
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The in situ ROS synthesis was measured using the oxidative fluorescent dye DHE (Sigma–Aldrich, Milwaukee, WI, USA) as previously described [4 (link)]. Thoracic aortas from all groups were embedded into an optimal cutting temperature (OCT) compound (O.C.T. Tissue-Tek, Sakura Finetek, Torrance, CA, USA) and then frozen in liquid nitrogen for cryostat sectioning. The frozen aortas were sliced into 5 μm thick sections, followed by incubation with DHE (2.5 μM) in a humidified light-protected chamber for 30 min at 37 °C. Images were examined using a confocal microscope (LSM 510 META, Carl Zeiss, Inc., Overkochen, Germany) with a 20× epifluorescence objective. Mean intensities are expressed as arbitrary densitometric units.
3
Histological Analysis of Rat Bladder and Prostate
4
Aortic Tissue Immunohistochemistry
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Aortic roots were embedded in OCT Tissue Tek (Sakura Finetek, Tokyo, Japan) for sectioning and staining with specific antibodies that are listed in the supplemental method section. Abdominal aortas were pinned for Oli-red O en face staining.
5
Femoral Tissue Preparation Techniques
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Femora from male newborn Cre and hKO mice were isolated and either fixed in 4% (w/v) paraformaldehyde overnight, embedded in paraffin and sectioned using a microtome (HM355 S, Thermo Fisher Scientific, USA) or embedded in O.C.T. Tissue-Tek (Sakura Finetek, Tokyo, Japan) and sectioned using a Leica Cryotome CM3050.
6
Multimodal Analysis of Lipid Droplets
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Tissues were rapidly frozen in melting isopentane after being coated in OCT Tissue-Tek (Sakura Finetek, The Netherlands). Serial 10 μm sections were cut at −20 °C on to SuperFrost Ultra Plus glass slides. Slides were fixed in Bouin's solution with 0.1% Triton X-100 (Sigma–Aldrich) for 30 min. Sections were stained for lipid droplets with freshly made, filtered oil red O (Sigma–Aldrich) made in 60% triethylphosphate (Sigma–Aldrich). β-galactosidase expression was identified using commercial reagents (MIR 2600, Mirus Bio, Madison, WI, USA). Sections were blocked in PBS with 1% BSA for 1 h then incubated overnight with PLIN5 antibody (Cat. no. GP31, Progen Biotechnik, Germany, ∼0.02 mg/ml) while mitochondrial staining was performed using the mitochondrial antibody directed against the oxidative phosphorylation complexes I–V (Total OXPHOS Cat. no. ab110413, Abcam, Cambridge, UK). For negative controls, primary antibodies were substituted for concentration matched guinea pig serum for PLIN5 (Antibodies Australia), or OXPHOS cocktail pre-absorbed 4:1 overnight with mouse IgG1 and IgG2a (Dako, Denmark). Immunohistochemistry images were analyzed for colocalization with ImageJ (NIH) by using Manders' colocalization coefficient, M2, which provides a fraction of the colocalizing objects in the images. Electron microscopy was performed as described previously in Ref. [34] (link).
7
Prostate Specimen Tissue Sectioning and Laser Capture Microdissection
8
Immunofluorescence of Mouse Eye Sections
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Mouse eyes were fixed in 4% PFA in 0.1 M phosphate buffer at 4°C for 30 min, a hole was created in the center of the cornea, and the eyes with a hole were fixed in the same 4% PFA solution at 4°C for additional 30 min. The fixed eyes were cryoprotected through an increasing gradient of sucrose in 0.1 M phosphate buffer (6.25% sucrose, on ice for 45 min; 12.5%, on ice for 45 min; and 25%, at 4°C overnight) followed by embedding in OCT Tissue-Tek (4583; Sakura Finetek USA) for cryostat. Immunofluorescence of frozen eye sections was performed as described previously (Masuda et al, 2014 (link)). Primary antibodies used are listed in Table S2. Secondary antibodies were anti-mouse, anti-rabbit or anti-goat IgG conjugated with Alexa Fluor 488, 549 or 647 (1:500; Invitrogen, Thermo Fisher Scientific). To observe signals clearer in the RPE, melanin pigment was bleached following the published protocol (Bhutto et al, 2004 (link)). Nuclei were stained with 4′,6-diamidino-2′-phenylindole dihydrochloride (DAPI, 10236276001; Roche) at room temperature for 10 min. The sections were mounted in a Fluorescent Mounting Medium (S3023; Dako), and images were acquired using an LSM 510 inverted laser scanning confocal microscope (Carl Zeiss).
Table S2.
9
Brain Tissue Preservation and Sectioning
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Fourteen days after MCAO, mice were deeply anesthetized with ketamine/xylazine (150 and 15 mg kg−1, respectively) and, upon complete loss of pedal reflexes, transcardially perfused with a 0.1 m PBS solution. Brains were carefully extracted and kept in 4% PFA in a 15 mL Falcon tube overnight at 4 °C. On the next day, for cryoprotection brains were incubated in 30% sucrose until they sank. Then, brains were frozen with the n‐butanol procedure in which 10 mL of n‐butanol were added in a 15 mL Falcon tube and cooled in liquid nitrogen to −50 °C. Once this temperature was reached, the brains were inserted into the Falcon tube, which was kept in liquid nitrogen for one minute to cool it to −80 °C. The brains were then ready for immediate storage at −80 °C. Deeply frozen brain tissue was mounted on the sliding microtome (Leica SM210R) with OCT Tissue‐Tek (Sakura Finetek Europe B.V., NL) and kept frozen with the addition of dry ice for cutting. Sequential sections of 60 µm in thickness were acquired and transferred sequentially into 96‐well plates filled with freezing medium (50% PBS, 25% glycerol, and 25% ethylene glycol) for further storage at −20 °C.
10
Prostate and Bladder Histology
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