Cardiac perfusion was performed with 20 ml PBS, and lungs were fixed in 4% paraformaldehyde (Sigma-Aldrich) for a week, dehydrated, and paraffin embedded. Sections (4 μm thick) were cut and stained with H&E, periodic acid–Schiff (PAS), and Masson’s trichrome (MTC) stains. To perform immunofluorescence staining, lung tissues were fixed with 2% paraformaldehyde in PBS with 30% sucrose at 4°C overnight and washed with PBS for 2 d at 4°C, then embedded in Optimum Cutting Temperature (OCT) compound (Sakura FineTek, Singapore); sections 5 μm thick were then cut on a cryostat (Leica, Singapore), air dried, and blocked with PBS containing 0.2% BSA. B cells were identified with rat anti-mouse B220 (1:200; eBioscience), T cells with Armenian hamster anti-mouse TcRβ (1:200; BD Pharmingen), and DCs with biotinylated Armenian hamster anti-CDllc (1:100; eBioscience) in PBS containing 1% normal mouse serum overnight at 4°C. Cy2-conjugated donkey anti-rat Ab (1:300; Jackson ImmunoResearch, West Grove, PA), Cy3-conjugated goat anti-hamster Ab, and donkey anti-avidin-labeled Ab (both 1:500; Jackson ImmunoResearch), respectively, were used for detection. Sections were counterstained with DAPI (KPL) and mounted with fluorescent mounting medium (Dako, Singapore) for analysis using a fluorescence microscope (AxioImager Z1, Zeiss, Singapore).
Optimum cutting temperature compound
Manufactured by Sakura Finetek
Sourced in United States, Japan, Singapore
Optimum cutting temperature compound is a medium-viscosity, water-soluble compound formulated to provide an optimal cutting temperature for sectioning frozen tissue samples. It is designed to facilitate the preparation of high-quality frozen sections for various applications in histology and pathology.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Fujifilm (4 mentions)
Cryostat, by Leica (3 mentions)
Paraformaldehyde (pfa), by Sakura Finetek (2 mentions)
Microm cryostat hm 560 mv, by Zeiss (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Axioskop2 upright fluorescence microscope, by Zeiss (2 mentions)
Cryostat microtome, by Leica (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Freezing microtome, by Leica (2 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Agilent Technologies (1 mentions)
Rat anti mouse b220, by Thermo Fisher Scientific (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Isopentane, by Merck Group (1 mentions)
Eclipse 80, by Nikon (1 mentions)
Typhoon phosphorimager, by Cytiva (1 mentions)
Nissl cresyl violet acetate, by Merck Group (1 mentions)
Iba1 antibody, by Fujifilm (1 mentions)
Hydrogen peroxide, by Sangon (1 mentions)
74 protocols using optimum cutting temperature compound
1
Histopathological and Immunofluorescence Analysis of Lung Tissues
2
Intestinal Tissue Harvesting and Freezing
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At necropsy, the entire section of ileum extending from the ileo-cecal valve through the distal flange was excised and then cut equally into proximal, mid- and distal sections. Tissues were rinsed with 0.15 M PBS and cut into multiple cross-sections for the culture of MAP and PCR to assess bacterial burden. Cross-sections immediately adjacent were processed for histopathology and IF labeling. A dry ice bath was prepared by combining 95% ethanol with dry ice and mixed until a slurry consistency was achieved. Isopentane (Sigma-Aldrich, St. Louis, MO, USA) was added to a tin cup and the cup was placed into the dry ice bath. The mid-ileal intestinal samples were washed with PBS, pH 7.4, and a cross-section was positioned luminal side down on a section of liver covered with Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) to protect the villi during the freezing process and to ascertain tissue orientation post-freezing. The intestine-liver sample was wrapped in foil and placed in the Isopentane for at least 5 min. The snap-frozen sample was transferred to dry ice for transport to storage at −80 °C, where it remained until tissue sectioning could be performed.
3
Assessing Residual DNA in Acellular Scaffolds
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Qualitative and quantitative assessments of residual DNA were evaluated against established criteria [15 (link), 16 (link)]: (1) the absence of visible nuclei in hematoxylin and eosin (H&E) or 4′,6-diamidino-2-phenylindole- (DAPI-) stained sections and (2) no DNA fragments exceeding 200 bp in length.
(1) Qualitative Assessment. Acellular scaffolds were embedded in Tissue-Tek Optimum Cutting Temperature (OCT) Compound (Sakura Finetek, USA), sectioned at a thickness of 10 μm, and captured on poly-L-lysine-coated slides. The tissues were then stained with H&E, and images were visualized using a light microscope (Nikon Eclipse-80, Japan).
(2) Quantitative Assessment. Acellular grafts were digested with 0.1 mg/mL proteinase K for 1 h at 55°C. The samples were then repeatedly extracted with phenol/chloroform and centrifuged to remove protein until the interface was free of white material. The aqueous phase extract was then mixed with 3 M sodium acetate and 100% ethanol and centrifuged to pellet DNA. The pellet DNA was rinsed with 70% ethanol, centrifuged, and dried. A 1.0% agarose gel containing ethidium bromide was then loaded with extracted DNA and electrophoresed for 2 h at 80 V, and the DNA was then visualized with ultraviolet transillumination.
(1) Qualitative Assessment. Acellular scaffolds were embedded in Tissue-Tek Optimum Cutting Temperature (OCT) Compound (Sakura Finetek, USA), sectioned at a thickness of 10 μm, and captured on poly-L-lysine-coated slides. The tissues were then stained with H&E, and images were visualized using a light microscope (Nikon Eclipse-80, Japan).
(2) Quantitative Assessment. Acellular grafts were digested with 0.1 mg/mL proteinase K for 1 h at 55°C. The samples were then repeatedly extracted with phenol/chloroform and centrifuged to remove protein until the interface was free of white material. The aqueous phase extract was then mixed with 3 M sodium acetate and 100% ethanol and centrifuged to pellet DNA. The pellet DNA was rinsed with 70% ethanol, centrifuged, and dried. A 1.0% agarose gel containing ethidium bromide was then loaded with extracted DNA and electrophoresed for 2 h at 80 V, and the DNA was then visualized with ultraviolet transillumination.
4
Mapping Brain and Spinal Cord Biodistribution
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Spinal cords and brain tissues from mice used for biodistribution were further analyzed through digital autoradiography. After perfusion and gamma counting, brain tissue was frozen in optimum cutting temperature compound (Sakura Finetek, Inc, Torrance, CA), and 20-µm-thick sections were cut using a cryostat microtome HM500 (Microm, Walldorf, Germany) at −20°C. Whole spinal cords and slides with brain slices were laid in a cassette and exposed to a 18F-sensitive storage phosphor film imaging plate (Fujifilm; GE Healthcare) for 20 hours at −20°C. Plates were scanned using a typhoon phosphorimager (Amersham Biosciences, Piscataway, NJ) and analyzed using ImageJ (image processing software, version 2.0.0). Brain section anatomy was confirmed by staining the exact same sections with Nissl (cresyl violet acetate; Sigma Aldrich, St. Louis, MO), as previously described.25 (link)
5
Immunohistochemical Analysis of Brain Tissue
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Immunohistochemistry assay was performed, as in our previous study (Zhang et al., 2017 (link)). Briefly, brain tissue samples were embedded in optimum cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and stored at −80°C. Samples sections were cut into 10-μm slices and antigen retrieval was performed using citrate buffer. Sections were treated with 3% hydrogen peroxide (Sangon Biotech Co., Ltd., Shanghai, China) in PBS for 10 min and then incubated in 5% BSA for 10 min. Sections were incubated overnight at 4°C with primary antibodies as follows: TH (F-11) antibody (1:50; Santa Cruz Biotechnology Inc.), nitrotyrosine (11C2) antibody (1:50; Santa Cruz Biotechnology Inc.), glial fibrillary acidic protein (GFAP) antibody (1:50; Santa Cruz Biotechnology Inc.) and ionized calcium-binding adapter molecule 1 (IBA-1) antibody (1:500; WAKO, Osaka, Japan). After washing 3 times with PBS for 5 min each, sections were incubated sequentially in HRP-conjugated goat anti-mouse and goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) for 2 h at 37°C. Sections were visualized with a 3,3-diaminobenzidine (DAB) peroxidase substrate kit (Boster, Wuhan, China). Integrated option density (IOD) was determined using an Image-Pro Plus 6.0 photogram analysis system (IPP 6.0; Media Cybernetics, Bethesda, MD, USA).
6
Mice Tissue Preparation for Immunofluorescence and RNA In Situ Hybridization
7
Immune Cell Profiling in Mouse Ear Tissue
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Mice were anesthetized with pentobarbital (Kyoritsu Seiyaku Co., Tokyo, Japan) and were perfusion-fixed with 4% paraformaldehyde. Ears were then removed and cryoembedded in optimum cutting temperature compound (Sakura Finetek, Tokyo, Japan), and 10-μm sections were cut using a cryostat. Cryosections were refixed in 4% paraformaldehyde for 15 minutes and were incubated for 1 hour at room temperature in PBS containing skin milk. After blocking, cryosections were incubated for 1 hour with primary biotinylated antibodies specific for Gr-1, CD11c, and F4/80 (1:50 dilutions; eBioscience). Cryosections were then incubated for 30 minutes with allophycocyanin-conjugated streptavidin (1:100 dilution; eBioscience) and nuclei were stained using Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan).
8
Quantifying Lung Metastasis in NSG Mice
9
Histological Assessment of Liver Pathology
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Paraffin‐embedded liver sections (5 μm) were deparaffinized and stained with hematoxylin and eosin (H&E) before mounting with mounting medium (Invitrogen). For Oil Red O staining, 6‐μm‐thick frozen sections were cut and stained with Oil Red O (Sigma). Digital images were captured on a light microscope (Olympus). To assess hepatic fibrosis, liver tissue samples were fixed with 2% paraformaldehyde in phosphate‐buffered saline (PBS) with 30% sucrose at 4 °C overnight, washed with PBS for 2 days at 4℃, and then embedded in optimum cutting temperature compound (Sakura FineTek, Singapore). Sections 5 μm thick were then cut on a cryostat (Leica, Singapore) and stained with Masson’s trichrome stains (Sigma‐Aldrich) using the standard protocol. Quantification of Masson’s trichrome staining was done by ImageJ software.
10
Aortic Valve Tissue Cryosectioning
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At the aforementioned time points, the animals were euthanized by cervical dislocation, and their hearts were extracted, washed thoroughly in cold, sterile PBS and frozen in optimum cutting temperature compound (Sakura Finetek USA Inc, Torrance, CA). Serial 5–8 µm transverse sections of the aortic valve were obtained using a Leica CM1860 cryotome (Buffalo grove, IL). Blood was collected in heparin coated tubes and plasma was stored at -80 freezer for further processing.
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