Formalin-fixed whole ear tissues were fixed in paraffin blocks and cut into 5 μm sections. Each section was stained with hematoxylin (Polysciences, Inc., Warminster, PA, USA) and eosin (Shimakyu Pure Chemicals, Osaka, Japan). Images were taken using a light microscope (Nikon Instruments Inc., Melville, NY, USA) to measure ear thickness. Digital micrographs were taken from representative areas at 200× fixed magnification. Ear thickness was determined using Axiovision 4.7 image analysis software (Carl Zeiss Vision GmbH, Munich, Germany).
Hematoxylin
Manufactured by Polysciences
Sourced in United States
Hematoxylin is a naturally occurring dye that is commonly used in histology and microscopy applications. It is a dark blue-purple stain that binds to nucleic acids, making it useful for staining cell nuclei. Hematoxylin is often used in combination with other stains, such as eosin, to provide contrast and enhance the visualization of cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Axiovision 4, by Zeiss (1 mentions)
Coverslip, by Thermo Fisher Scientific (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Eosin y, by Merck Group (1 mentions)
Isopentane, by Merck Group (1 mentions)
Eclipse 50i microscope, by Nikon (1 mentions)
Entellan mounting medium, by Electron Microscopy Sciences (1 mentions)
Dm3000, by Leica (1 mentions)
5 protocols using hematoxylin
1
Histological Assessment of Ear Thickness
2
Tissue Staining and Mounting Protocol
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Bake slides for 1 hour at 60°C (
Deparaffinize and rehydrate sections: 3 × 5 min Xylene, 2 × 5 min 100% ethanol, 1 × 3 min 95% ethanol, 1 × 3 min 70% ethanol, 1 × 5 min ddH2O.
Hematoxylin (Polysciences, Inc.) stain for 1 min 15 s, and rinse in ddH2O 3 times.
Place in bluing reagent (Epredia) for 1 min, and rinse in ddH2O 3 times.
Eosin (Epredia) staining and dehydration: 1 × 30 s Eosin, 3 × 2 min 95% ethanol, 3 × 2 min 100% ethanol, 3 × 5 min Xylene.
Coverslip (Fisher) slides using Xylene-based mounting medium (Vector).
3
Hematoxylin-Eosin Tissue Staining
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Sections were deparaffinized and rehydrated, and nuclei were stained with hematoxylin (Polysciences, Warrington, PA, USA). Sections were rinsed in water and then placed briefly in eosin Y (Sigma-Aldrich, St Louis, MO, USA). Sections were dehydrated and mounted using Permount (Fisher Scientific, Waltham, MA, USA).
4
Analyzing Renal Artery Morphology
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SRA segments were collected at the end of experiments into optimal cutting compound (ProSciTech), frozen in cold isopentane (Sigma) and stored in −80 °C until required. The renal arteries were used as a landmark to position the tissue and serial sections were taken from the renal arteries to the region of maximal dilatation. Five SRAs were randomly selected from the vehicle, fondaparinux, and dabigatran groups using an online random number generator (https://www.random.org/ ). Four serial sections of 6 μM thickness were placed on poly-L-lysine coated slides, air dried and subsequently fixed in acetone for 10 min at −20 °C. Adjacent sections were stained with hematoxylin (Polysciences Inc., #24245) and eosin (Polysciences Inc., #09859) (H&E) and mounted in Entellan mounting medium (Electron Microscopy Sciences, #14800) to assess gross morphology. Verhoeff Van Giessen (VVG) staining (Polysciences Inc., #25089) was performed to assess the integrity of SRA elastin. Sections stained with H&E and VVG were photographed using a Nikon Eclipse 50i microscope fitted with a CCD Camera (DSFi1). Digital images were captured to a PC supported with NIS Elements (version F2.30) and analysed as previously described42 (link). Histological evaluations were carried out in a blinded fashion in 3–4 sections per sample (mean coefficient of variation (CV) = 2.97%; n = 5).
5
Histological Analysis of Excised Lungs
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Excised lungs were fixed overnight in 4% pformaldehyde for 24 h, washed in PBS and embedded in paraffin for H&E staining: 4µm tissues sections were stained with Hematoxylin (Polyscience, Inc Warrington, PA) for 40'' and with Eosin (Sigma, St. Louis, MO, USA) for 30''. Tissues sections were examined under a light microscope Leica DM3000 (Leica, Wetzlar, Germania).
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