Livers were fixed in 4% paraformaldehyde (Polysciences, Inc., Warrington, PA) and embedded in paraffin or Tissue-Tek® Optimum Cutting Temperature (OCT) Sakura Finetek, Torrance, CA) for sectioning. Immunofluorescence (IF) staining signals for PROM1 (1:50 dilution, eBiosciences, San Diego, CA), CYTOKERATIN-19 (1:100 dilution, gift from JR Friedman, University of Pennsylvania, Philadelphia. PA), and LacZ (1:400, Bioss, Woburn, MA) were detected by secondary antibodies conjugated either with anti-mouse cyanine (Cy) 3, Cy5, anti-rat Cy3/Cy5, or anti-rabbit Cy3/Cy5 (1:200 dilution, Jackson ImmunoResearch Labs, West Grove, PA) (9 (link), 10 (link), 20 (link)). Images were then acquired by a Leica DM5500B IF microscope using Leica Suite Advanced Fluorescence (LAS AF) 6000 software (Leica Microsystems, Wetzlar, Germany). Immunohistochemistry was performed using 2.1 μg/mL anti-INTEGRIN-β6 (gift from Shelia Violette, Biogen, Inc). Bright-field images were captured using a Leica DM1000 (DFC290) transmitted light microscope (Leica Microsystems AG, Heerbrugg, Switzerland) with Leica Application Suite (version 2.7.1R1).
Tissue tek optimum cutting temperature
Manufactured by Sakura Finetek
Sourced in United States, Japan
The Tissue-Tek optimum cutting temperature (OCT) compound is a water-soluble, glycol-based embedding medium used for frozen section preparation of biological specimens. It is designed to provide optimal cutting characteristics for cryosectioning.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Polysciences (1 mentions)
Anti rat cy3 cy5, by Jackson ImmunoResearch (1 mentions)
Anti rabbit cy3 cy5, by Jackson ImmunoResearch (1 mentions)
Dm5500b if microscope, by Leica (1 mentions)
Prom1, by Thermo Fisher Scientific (1 mentions)
α sma, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Senescence β gal staining kit, by Cell Signaling Technology (1 mentions)
Digital sight ds u2 camera, by Nikon (1 mentions)
Eclipse te300 inverted microscope, by Nikon (1 mentions)
Cm1510, by Leica (1 mentions)
Hematoxylin solution, by Thermo Fisher Scientific (1 mentions)
Eosin solution, by Thermo Fisher Scientific (1 mentions)
Balb c nude mice, by Orient Bio (1 mentions)
7 protocols using tissue tek optimum cutting temperature
1
Immunofluorescence and Immunohistochemistry of Liver
2
Immunohistochemical Analysis of Fibrotic Markers
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One quarter of muscle was also fixed in 4% PFA overnight, gradually dehydrated through 15% and 30% sucrose in phosphatebuffered saline (PBS), and embedded in Tissue-Tek Optimum Cutting Temperature (OCT, Sakura Finetek Inc., Torrance, CA, USA). Cryosections (8 µm) were blocked in 20% normal goat serum in 0.5% Triton X-100/PBS for 15 min, and then incubated overnight at 4°C with primary antibody against TGF-β1 at 1:200 dilution (R&D Biotechnology, Minneapolis, MN), CTGF at 1:200 (R&D Biotechnology) and α-SMA at 1:100 dilution (Sigma Chemical, St. Louis, MO). After washing, sections were incubated with secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1h at 37°C, and counterstained with DAPI (Sigma Aldrich Inc., St. Louis, MO). Sections incubated with PBS instead of the primary antibodies were used as negative controls. Images were captured with Nikon Eclipse 600 fluorescence microscope (Tokyo, Japan) at a magnification of ×40, and the positive cells visible in the photomicrograph were counted in five random vision fields for three sections of each case.
3
Histological Analysis of Micromass Cultures
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The induced micromasses were frozen in Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan), sliced into 5-μm-thick sections, and fixed in 10% formalin solution (Wako, Osaka, Japan) for 10 min. Each section was then rinsed with deionized water and stained with 6% Safranin O solution (Chroma, Münster, Germany) for 5 min at RT. The sections were then rinsed with deionized water, counterstained with hematoxylin for 2 min, and observed using a microscope.
4
Senescence Assay for PDX Tumors
5
Quantitative Analysis of Hepatic Steatosis
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Liver samples were included in Tissue-Tek® optimum cutting temperature (OCT) compound (Sakura Finetek, Alphen aan den Rijn, The Netherlands), and later in liquid nitrogen for freezing. Each liver block was serially sectioned (7 μm) with a cryostat (CM1510 S, Leica, Wetzlar, Germany) to perform the H&E and Oil Red O stains using standard techniques. All liver sections measured >1.5 cm in length and showed more than ten complete portal tracts. The percentage of hepatocytes containing lipid droplets was determined for each 10× field. An average percentage of steatosis was then determined for the entire specimen. Images of sections of both H&E and Oil Red O were acquired using an inverted Eclipse TE300 microscope (Nikon, Tokyo, Japan) coupled to a Digital Sight DS-U2 camera (Nikon). For images of Oil Red O stains, quantification was performed using IP Win32 v4.5 software (Acromag).
6
Hematoxylin and Eosin Staining of EDL Muscles
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This staining was used to determine the extent of any fiber damage in EDL muscles. Hematoxylin and eosin staining staining was carried as previously described (Thabet et al. 2005 ). Briefly, muscles were embedded in Tissue‐Tek® Optimum Cutting Temperature (Sakura Finetek, Torrance, CA) compound and frozen in isopentane precooled in liquid nitrogen. Ten‐μm‐thick cross sections were cut from the mid‐belly of each muscle using a cryostat (Leica Microtome, HM 500M, Concord, ON, Canada) and mounted on Superfrost Plus slides. Cross sections were incubated in hematoxylin solution (Shandon), washed dipped in eosin solution (Shandon, Thermo Scientific), mounted in Permount before the coverslip was placed over the sample.
7
Subcutaneous Tumor Xenograft Assay
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Animal studies were approved by the Institutional Animal Care and Use Committee at Catholic University of Korea. Hep‐2 cells were pre‐treated with control or 14‐3‐3ζ siRNA for 48 hours, after which 1 × 107 cells in 200 μL PBS were injected subcutaneously into the flanks of 5‐6‐week‐old male BALB/c nude mice (Orient bio Inc). Two weeks after tumour cell inoculation, all mice were sacrificed, and individual tumours were weighted and fixed in 4% paraformaldehyde and embedded in paraffin or frozen in Tissue‐Tek optimum cutting temperature (Sakura Finetek). Staining for 14‐3‐3ζ and p27 was carried out on paraffinized sections.
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