Mice were euthanized by carbon dioxide inhalation followed by cervical dislocation as per standard operating procedure approved by IACUC of The Ohio State University. Femurs were collected, fixed in formalin for at least 24 h followed by decalcification for another 48 h in Leica Surgipath decalcifier. These bones were then sectioned longitudinally for H&E staining. The megakaryocytes identified by their characteristic shape of a large lobulated nucleus41 (link), were counted in an area of 2.5 mm2 with the use of Image Scope v12.2.2.5015 (www.aperio.com ) and confirmed by board certified histopathologist. In brief, quantification commenced 125 µm from the growth plate and an area of 2500 µm was scored.
Surgipath decalcifier
Manufactured by Leica
The Surgipath decalcifier is a laboratory equipment designed to remove calcium deposits from tissue samples. It is used to soften and prepare hard tissue specimens, such as bone or calcified samples, for subsequent histological processing and analysis.
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2 protocols using surgipath decalcifier
1
Quantification of Megakaryocytes in Murine Bone
2
Histological Assessment of Zebrafish and Medaka
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Eight samples for histological assessment were collected from control and each treatment at 5 dpf for zebrafish and at 14 dpf for medaka. The larvae were anesthetized with 0.01% MS 222 (Tricaine Methanesulfonate), and then immediately fixed in the Surgipath Decalcifier (Leica) for 24 h at 4°C. After dehydration in ethan ol, all specimens were embedded in paraffin (Bio-Optica, Italy) and sectioned at a thickness of 5 µm with a rotary automatic
microtome (Leica Microsystems, Wetzlar, Germany). Serial sections were stained with hematoxylin and eosin (Bio-Optica, Isttaly), and examined with a motorized Zeiss Axio Imager Z1 light microscope (Carl Zeiss AG, Werk Göttingen, Germany), equipped with an AxioCam digital camera (Zeiss, Jena, Germany) for the acquisition of images. This protocol was already published (Fasulo et al. 2010 , Maisano et al. 2016 , Maisano et al. 2017)
microtome (Leica Microsystems, Wetzlar, Germany). Serial sections were stained with hematoxylin and eosin (Bio-Optica, Isttaly), and examined with a motorized Zeiss Axio Imager Z1 light microscope (Carl Zeiss AG, Werk Göttingen, Germany), equipped with an AxioCam digital camera (Zeiss, Jena, Germany) for the acquisition of images. This protocol was already published (Fasulo et al. 2010 , Maisano et al. 2016 , Maisano et al. 2017)
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