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After washing with Sodium Cacodylate buffer, samples were fixed by 2% Osmium (Electron Microscopy Sciences) for 15 min. The samples were dehydrated in increasing percentages of anhydrous ethyl alcohol (50%, 75 %, 90 % and 100.
Following one wash in labeling buffer, samples were fixed using Cytofix Solution according to manufacturer's directions (BD), resuspended in 300 µl labeling buffer, and data were collected on a FACSCalibur (BD).
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All samples were fixed 10% buffered formalin and embedded in paraffin.
The samples were fixed with 4% buffer neutralised formalin, paraffin-embedded and cut into 4 μm sections.
Biopsies were taken from the iliac crest of adult sheep (∼10 years old; approved by Danish Animal Experiments and Inspectorates, 2011/561-1959) from the study described in Andreasen et al. The bone samples were fixed in 4% buffered formaldehyde for 24 h, before transferring to 0.1% buffered formaldehyde.
Lung samples were fixed in 10% buffered formalin.
The samples were fixed in 10% buffered-formalin for 48 hours prior to paraffin embedding.
Then these samples were fixed in 10% buffered formalin and embedded in paraffin.
Tissue samples were fixed in 10% buffered formalin, processed routinely, embedded in liquid paraffin and sectioned at 3 µm.
Brain samples were fixed in 10% buffered formalin, dehydrated, and embedded in paraffin wax for examination for pathology.
The heart tissue samples were fixed in phosphate buffered 4% paraformaldehyde and embedded in paraffin, and 4 µm sections were stained with haematoxylin-eosin according to standard protocols.
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