Nis elements f2

Samples were deparaffinized and treated with 10% hydrogen peroxide and pepsin (EXIQON), and hybridized with DIG‐labeled LNA probes (EXIQON) complementary to miR‐6769b‐3p or miR‐499a‐3p. After being blocked with 10% normal goat serum and 5% BSA in PBS, specimens were incubated with anti‐DIG Fab fragments (Roche) and secondary antibody conjugated with alkaline phosphatase (Invitrogen) using nitroblue tetrazolium/5‐bromo‐4‐chloro‐3‐indolyl phosphate (NBT‐BCIP) as substrate. Counterstaining was conducted using a nuclear fast red solution (Vector Laboratories). Nikon 80i microscope and Nikon NIS‐Elements F 2.3 software (Nikon) were used for analysis. microRNAs expression was assessed on the basis of the intensity and the scale of staining using Image‐Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA).

Histomorphometric analysis was performed with a semi-automated digitizing image analyzer system, consisting of an Eclipse E600 stereoscopic microscope, a computer-coupled Digital Eclipse DXM 1200 digital camera and NIS-Elements F 2.20 imaging software (Nikon Corporation, Tokyo, Japan) (2 (link)). Bone static indices included the following: Bone-to-implant contact (BIC, %) = summation of the lengths of direct bone-to-implant contact interface/the length of total implant surface; peri-implant bone fraction (BF, %) = the percentage area of the bone within the rectangular region 2.0 mm from the axis of the implant; and thickness of the bone lamellae (TBL, μm) in direct contact with the implant at cancellous bone area (Fig. 2). For evaluation of the TBL, five equally distributed sites were chosen for each screw and the mean value of all screws was accepted as the value of the index of the section (7 (link)). Three discontinuous sections of each specimen were measured at a magnification of ×100 and the mean of the three sections was accepted as the value of the specimen for each index.

Cell growth of LLC-PK1 and HeLa cells was observed and recorded by a confocal microscope (Eclipse TE300, Nikon, Tokyo, Japan) at 3 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h. Images were acquired with the software NIS-Elements F 2.20 (Nikon, Tokyo, Japan).

HeLa cells were transfected with pcDNA3/RSU1P2 or the control. At 48 h after transfection, the cells were washed by PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. After incubation in PBS for 5 min, the cells were permeabilized for 2 min with 0.1% (v/v) Triton X-100 on ice. The samples were washed twice with PBS for 5 min each, incubated for 1 h in mixed solution (enzyme solution:label solution = 1:9, Roche) and protected from light. Before staining with DAPI (4, 6-diamidino-2-phenylindole, 300 nmol/L from Invitrogen), the cells were washed in PBS three times, 5 min each. After the final wash, the samples were visualized with an imaging system (NIS Elements F 2.20 imaging software, Nikon, Tokyo, Japan).

Osteoclast resorption activity was performed using hydroxyapatite substrate coated culture plates. Cells were seeded at a concentration of 4 × 10⁴ cells/well in 24-well Osteoclast Activity Assay Substrate plates (OAAS, OCT USA, Inc., Irvine, CA). Cultures were treated with BMP2/7 (5 or 50 ng/ml) and rhRANKL (50 ng/ml) ± ATRA (1 μΜ) for 7 days. Cultures were washed with 6% sodium hypochlorite solution to remove the cells. The wells were examined in a Nikon microscope with NIS-Elements F2.20 and photographed in color at a final magnification of 100×. Approximately 24 photomicrographs were collected per well using a systematic random-sampling strategy. The photomicrographs were printed in color for histomorphometric analysis. The resorption areas that exhibited white color on the pale brown background were measured using the point-counting technique [44 (link)].

Five-micrometer thick sections from formalin fixed and paraffin-embedded kidneys were stained with 1:1200 diluted rabbit anti-ODCrp[A] antibody and with its preimmune serum as control using Vectastain Elite ABC Kit (Vector Laboratories Inc., Burlingame, U.S.A.) according to the manufacturer’s instructions essentially as described [29 (link)]. Light microscope photographs were taken with an Olympus BX51 microscope (Olympus Optical, Tokyo, Japan) and a Nikon Digital Sight DS-5M camera (Nikon Corporation, Tokyo, Japan) using NIS-Elements F2.30 software (Nikon Corporation). Digital image processing was performed with PhotoScape v3.6.1 (Mooii Tech, Informer Technologies Inc., Los Angeles, U.S.A.).

After a faint counterstaining with hematoxylin (Merck, Darmstadt, Germany), the specimens were dehydrated using alcoholic solutions with a progressively increased alcohol concentration, cleared in xylene (Sigma Aldrich, Milan, Italy), and mounted with DPX (Sigma Aldrich, Milan, Italy). All samples were examined using an Olympus BX40F4 microscope (Olympus Official, Tokyo, Japan) equipped with a Nikon Digital Sight DS-Fi1 camera (Nikon Corporation, Tokyo, Japan). Images were acquired with NIS-Elements F2.30 software (Nikon Corporation, Tokyo, Japan).