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The resected specimens were then decalcified, trimmed, and embedded in paraffin. The blocks were sectioned serially in 5 μm thickness perpendicular to the long axis of the socket. The central-most section was chosen for histological and histomorphometric analyses. Hematoxylin/eosin and Masson's trichrome staining were performed. The histological slides were scanned using digital slide scanner (Panoramic 250 Flash III, 3DHISTECH, Budapest, Hungary) and observed through CaseViewer (version 2.1, 3DHISTECH). The histomorphometric analysis was performed using CaseViewer (version 2.1, 3DHISTECH) and Photoshop CS6 (Adobe, CA, USA) by a single experienced investigator (H.C.L.) who was blinded to the group assignment.
The histomorphometric measurements were performed for both the entire augmented area and three rectangular regions of interest (ROIs) within the augmented area (each of size 2.0 mm²) set up by dividing the entire augmented area into three equal areas, defined as the coronal_1/3, middle_1/3, and apical_1/3 areas. The following parameters were measured (Figure 2): (i) total augmented area including new bone, residual material, and nonmineralized tissue (TA), (ii) area of newly formed bone (NB), and (iii) area of residual bone substitute material (RM). The number of blood vessels (BV) was measured in each ROI.

After fixation in 4% paraformaldehyde, testicular tissues were dehydrated with graded 75, 85, 95, and 100% alcohol, infiltrated in xylene, embedded in paraffin blocks, sectioned into 4 μm-thick sections, mounted onto slides, and stained with hematoxylin and eosin (HE). The section images were acquired on a Pannoramic MIDI scanner (3DHISTECH, Hungary). Perimeters and cross-sectional areas of seminiferous tubules (μm and μm²) were measured using the liner measurement annotation of Caseviewer (3DHISTECH, Hungary). Thicknesses of spermatogenic cell layers (μm) were measured using liner measurement annotation of Caseviewer (3DHISTECH, Hungary). The thicknesses of each seminiferous tubule were measured 4 times and then averaged. On average, 40–50 tubules were analysed per rabbit.

Cells were treated with or without AZD1775 (1μM). Cytochalasin B (3 μg/mL) was added at the 20th hour to each culture and incubated at 37^◦C. After 24hrs, the cells were centrifuged at 1000 rpm for 5 min. The supernatant was removed, and the pellet was treated with weak hypotonic solution (0.075 M KCl/0.9% Saline, 1:9) and incubated at 37^◦C for 5 min. After this, the cells were centrifuged and the pellets were fixed in fresh fixative (methanol:acetic acid, 3:1). Cells were dropped onto glass slides prepared and stained with ProLong Gold Antifade Mountant with DAPI (CST) for scoring. The slides were scanned with a 20x/0.8NA plan-apochromat objective in a 3DHistech Pannoramic 250 Flash Scanner (3DHISTECH, Öv u. 3., Hungary). The scanned images were evaluated with Case Viewer (3DHISTECH) in the setting of 20x in Case Viewer. At least 10 areas following the standard specifications, were scored for each slide.