The mice were euthanized via cervical dislocation six weeks after the aortic-valve injury. Hearts were isolated and blood was flushed by injecting 0.9% sodium chloride solution into the LV. Afterwards, the collected hearts were embedded in a tissue-freezing medium and sectioned (8 μm thickness). The sections were stained with hematoxylin and eosin according to standard protocols. Tissue calcification was measured using von-Kossa staining (Abcam Staining Kit). Collagen was visualized using Pico-Sirius-Red staining (Sigma Aldrich). Images were obtained using light/polarization (Sirius-Red) microscopy at 10x (Axio Observer, Zeiss, Germany). Aortic valve area, collagen, and calcium deposits were measured with Zeiss ZEN Imaging Software.
Picosirius red
Manufactured by Merck Group
Picosirius red is a laboratory reagent used for the quantitative analysis of collagen content in biological samples. It binds specifically to collagen fibers and can be used in a variety of applications, including histological staining and spectrophotometric assays.
Automatically generated - may contain errors
Lab products found in correlation
Dm4500, by Leica (2 mentions)
Zen imaging software, by Zeiss (1 mentions)
Axio observer, by Zeiss (1 mentions)
Sirius red, by Merck Group (1 mentions)
Saturated aqueous picric acid, by Merck Group (1 mentions)
Axio lab a1 microscope, by Zeiss (1 mentions)
Axiocam icc1, by Zeiss (1 mentions)
Entellan, by Merck Group (1 mentions)
Picric acid, by Merck Group (1 mentions)
Masson s trichrome, by Merck Group (1 mentions)
6 protocols using picosirius red
1
Aortic Valve Injury Histological Analysis
2
Histological Staining Protocols
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Reagents for Acid Fuchsin Orange G and Picosirius red staining were purchased from sigma and performed according to standard procedures (ihcworld.com). Picosirius red analysis was performed on a Leica DM4500 using a polarized filter set at maximal contrast.
3
Acid Fuchsin Orange G and Picosirius Red Staining
4
Picrosirius Red Staining of GRMD Samples
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Frozen OCT‐embedded GRMD samples were sectioned at 10 µm, and PicoSirius red staining was performed as previously described.12 Briefly, sections were fixed in 4% paraformaldehyde for 10 minutes, rinsed, air dried, stained for 1 hour in 0.1% (wt/vol) Sirius red (Sigma‐Aldrich), dissolved in saturated aqueous picric acid (Sigma‐Aldrich), washed in 2 changes of 0.5% acetic acid, dehydrated in 3 changes of 100% ethanol, cleared with Citra Solv, and mounted with Cytoseal. The resulting slides were viewed under circularly polarized light scope (DMLP; Leica), rotating polarizer, rotating analyzer, and dual quarter wave plates on a camera system (Micropublisher 5.0; Q Imaging).
5
Picrosirius Red Staining for Collagen
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Frozen lung sections were incubated in picrosirius red solution (0, 2 gr of Picosirius Red diluted in 100 ml of 1, 2% picric acid, both Sigma-Aldrich), for one hour. After washing with water, tissue sections were stained with hematoxylin for 5–10 seconds. Slides were washed with running tap water and dehydrated in 70%, 90% and absolute ethanol, followed by xylene. Entellan (Merck) was used to mount the coverslip. Images were obtained using Axio Lab.A1 microscope (Zeiss) with 200 × magnification and AxioCam ICc1 (Zeiss). Collagen quantification was made with ImageJ.
6
Histological Analysis of Mouse Hearts
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Hearts from adult mice were fixed by Langendorff perfusion or by transcardiac perfusion using 4 % paraformaldehyde (PFA) and embedded in paraffin. Fetal and neonatal hearts were isolated and then fixed by submersion in 4 % PFA before embedding. From embedded hearts, we obtained 5 mm transverse sections for analysis. Masson’s Trichrome (Sigma Aldrich, HT15, St. Louis Missouri) and Picosirius Red (Sigma Aldrich, 365548) staining were according to supplier’s instructions. Fiber diameter index was quantitated using Hamamatsu-NDP software.
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