Bz 9000 digital microscope

Immediately following dissection, the right femur containing the implants was kept in cold saline and scanned by a microcomputed tomography (CT) system (SkyScan1275, Bruker, Billerica, MA, USA) at 90 kV and 40 mA to investigate the effects of the newly formed bone around the implants. The bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th) within the regions of interest (ROI; 500 μm around the implant and 2 mm below the epiphyseal line) in the CT images were determined using Morphometric software (CTAn; Bruker, Billerica, MA, USA).
After micro-CT scanning, the specimens were fixed in 70% ethanol solution for 3 d and subsequently stained with hematoxylin and eosin (H&E). Sections were analyzed by histomorphometry using a BZ-9000 digital microscope (Keyence Co., Osaka, Japan).

For cell attachment measurement, the CellSaic was fixed with 4% phosphate paraformaldehyde buffer, and frozen thin sections (thickness 5 µm) were prepared. After washing with PBS, the cells were permeabilized with 0.1% Triton X-100, and DAPI-Fluoromount-G (SouthernBiotech, Birmingham, AL, USA) was used for nuclear staining.
For the determination of calcium deposition, the CellSaic was cultured in osteogenic medium for 7, 14, and 21 days, during which frozen thin sections (thickness of 5 µm) were prepared, and 2% Alizarin Red S solution (Sigma-Aldrich, St. Louis, MO, USA) was used to stain calcium deposits in the extracellular matrix of BMSC-CellSaic and DPSC-CellSaic for 10 min at room temperature, and then washed with PBS. A BZ-9000 digital microscope (Keyence Co., Osaka, Japan) was used to capture all the images. The experiment was repeated three times.

All samples were decalcified using Decalcifying Solution B (Cat. No.: LEP2494; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) and then embedded in paraffin. Paraffin sections of 4-µm thickness were obtained by sectioning with KAC Co., Ltd. (Kyoto, Japan) and then subjected to HE and TRAP staining. Staining was performed according to standard protocols at KAC Co., Ltd. HE and TRAP slides were examined with a BZ-9000 digital microscope (Keyence Corporation, Osaka, Japan).

Four-micrometer-thick non-decalcified frozen sections were obtained from the fixed samples utilizing the Kawamoto method [30 (link)]. Thin sections were then processed via hematoxylin-eosin staining. Images were captured utilizing a BZ-9000 digital microscope (Keyence Co., Osaka, Japan). Histomorphometric analysis was conducted to calculate the thickness of tissues above the original bone level in the calvaria defect areas utilizing Adobe Photoshop Elements (Adobe Systems Inc., San Jose, CA, USA) and ImageJ (Image J 1.50i; NIH, Bethesda, MD, USA). This process was conducted as follows: (1) capture images by utilizing the BZ-9000 digital microscope, (2) draw a continuous curve along each outer side of the bone beds from end to end to mark the original bone surface utilizing Adobe Photoshop Elements, and (3) measure the distances from the margin vertexes of tissue to the original bone surfaces marked in (2) utilizing ImageJ.

After performing micro-computed tomography, the femoral specimens were collected and stained using the Villanueva method to assess osseointegration [50 (link)]. All sectioning procedures were executed under running water to maintain a cool temperature. A BZ-9000 digital microscope (Keyence Co., Osaka, Japan) and laser scanning microscope (Carl Zeiss, Oberkochen, Germany) were used to analyze the ingrowth of the bone. The excitation/emission wavelengths of the chelating fluorochromes were 351/460 nm for oxytetracycline hydrochloride (blue), 543/617 nm alizarin red S (red), and 488/517 nm for calcein (green), respectively. The bone area ratio (BA), bone-implant contact (BIC), and labeled bone area (LBA) were assessed using ImageJ in a 200× field around the implant and in a section 2 mm below the growth plate for each.

To analyze the radiopacity and morphology of the newly formed bone in the defects, the treated mandibles were assessed by micro-computed tomography (µCT) scanning (SMX-130CT; Shimadzu) at 71 μA of 75 kV radiation. Images were saved at a resolution of 512 × 512 pixels. TRI/3D-Bon (Ratoc System Engineering, Tokyo, Japan) was used to reconstruct the anterior and the diagonally upward views of the mandible. Bone mineral density representing calcified bone tissue was assessed using cylindrical phantoms containing hydroxyapatite (hydroxyapatite content: 200–1550 mg/cm³). The BV/TV, bone mineral content (BMC)/TV, and BMC/BV were calculated to assess the mineralized tissue volume, weight, and density in the defects. For the histological evaluation, each calvaria was fixed with 4% paraformaldehyde phosphate buffer solution, decalcified with formic acid, dehydrated, and embedded in paraffin. Thin sections (5 μm in thickness) were prepared and stained with hematoxylin–eosin and toluidine blue. All images were captured using the BZ-9000 digital microscope (Keyence Co., Osaka, Japan).

Male mice (4 WT and 4 DBZ KO) aged 12 weeks were used. Brains were removed and Golgi-Cox staining was performed using an FD Rapid GolgiStain Kit (FD NeuroTechnologies, Ellicott City, MD, USA) according to the manufacturer’s instructions. Unfixed brain samples were processed as previously reported (Koyama et al., 2013a (link)). The coronal brain blocks were immersed in a solution of equal parts Solution A and B at room temperature for 2 weeks and then soaked in Solution C at 4°C for 48 h. After freezing with dry-ice powder, the brain samples were sliced into 250 μm pieces at −22°C using a cryostat microtome. Each frozen section was mounted with Solution C on a 0.5% gelatin-coated glass slide. Slides were allowed to dry naturally at room temperature and were then stained with a mixture (Solution D: Solution E: DW; 1:1:2) for 5 min, after which the slides were rinsed in distilled water twice for 4 min each. Slides were dehydrated in an ascending ethanol series and then sealed with Entellan (Merck, Darmstadt, Germany) through xylene. The preparations were observed in detail with a BZ-9000 digital microscope (Keyence Corporation, Osaka, Japan).