Histological Analysis of Root Resorption

Sections were deparaffinized at 60 °C for 30 min and transferred to xylene (9713.2, Carl Roth GmbH, Karlsruhe, Germany) for 20 min. They were hydrogenated by a descending alcohol series followed by staining with Mayer hematoxylin solution (1.07961.0500, Merck KG, Darmstadt, Germany) for 10 min. After incubation under running warm water for 5 min, slides were counterstained with eosin G solution 0.5% (X883.2, Carl Roth GmbH, Karlsruhe, Germany) for one minute. After rinsing the samples under warm tap water, they were dehydrated by an ascending series of alcohol. After incubation for 20 min in xylene, the coverslips were applied with entellan (1.07961.0500, Merck KGaA, Darmstadt, Germany). The stained histological sections (Figure 2b) were digitized under the microscope (Olympus IX50 microscope in combination with DP2-SAL camera, Olympus, Hamburg, Germany) and evaluation of the relative extent of root resorption area at the distobuccal root of the upper first molar in relation to the respective total root area within the same slice was performed with ImageJ (Ver.147, National Institutes of Health, Bethesda, MD, USA), as described in Kirschneck et al. [25 (link),27 (link),38 (link)].