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. 2016 Aug 3;7:330. doi: 10.3389/fphys.2016.00330

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Copyright © 2016 Ley, Romantsik, Vallius, Sveinsdóttir, Sveinsdóttir, Agyemang, Baumgarten, Mörgelin, Lutay, Bruschettini, Holmqvist and Gram.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Hb immunolabeling within the brain following preterm rabbit pup IVH. The presence of Hb following IVH was characterized by Hb (displayed as red) immunolabeling of selected regions of interests (ROI) of peroxidase activity corresponding areas, recorded in parallel sections. Rabbit pups with IVH or sham controls were euthanized at 72 h of age followed by saline and freshly prepared 4% PFA perfusion. Afterwards the brains were dissected out from the skulls and immersed in 4% PFA. Brains were prepared and sections at the levels of rostral forebrain (Level 1) and caudal midbrain (Level 4), were immunolabeled for Hb as described in the Materials and Methods Section. Fluorescence microscope analyses were performed on a wide-field Olympus microscope (IX73) and slide scanning were performed on a Hamamatsu NanoZoomer 2.0-HT Digital slide scanner: C10730. Scanning was performed with a 40 × magnification lens. Images used for illustrations, from ROI's, were grabbed with the viewer software NDP.view2 Viewing software. DAPI labeled cell nuclei are blue. Scale bar of slide scan images indicate 2.5 mm and of ROI images indicate 25 μm.