Nis elements d imaging software

Mice were deeply anesthetized via Ketamine/Xylazine (120/5 mg/kg) intraperitoneal (IP) injection and fixed by cardiac perfusion using a 0.2% glutaraldehyde, 4% paraformaldehyde (PFA) solution. Brain, ribcage, lymph nodes, salivary glands, thymus, heart, lung, liver, spleen, stomach, kidney, intestine, urogenital, muscle, and hind limb tissues were dissected, rinsed in phosphate buffered saline (PBS) and post-fixed for 30 minutes in a 0.2% glutaraldehyde, 4% PFA solution. Tissues were washed and incubated in X-gal (1 mg/mL) staining solution for roughly 12 hours at 37 °C. After staining, tissues were washed, post-fixed in 4% PFA and cleared in a series of 50%, 70% and 100% glycerol. Photographs were taken with a Nikon SMZ1500 stereomicroscope and Nikon DS-Ri1 digital camera using NIS-Elements D Imaging Software (Nikon).

Actin staining: fixation and permeabilization was performed with 4% paraformaldehyde and 0.2% Triton X-100 for 30 min at + 4°C. Cells were treated with 50 µg/ml solution of Alexa Fluor 594-phalloidin (Sigma-Aldrich) for 40 min.
Occludin staining: cells were fixed by adding 100% methanol for 10 min at room temperature and probed with anti-occludin antibody (Abcam ab59720) over night at +4°C. Bound antibody was detected with Alexa fluor 488 conjugated anti-rabbit IgG antibody (Invitrogen, A21206).
Immunofluorescence assay for NF-kB: Caco-2 cells were grown on the chambered cover glass for 3 days, fixed with 4% buffered paraformaldehyde (pH 7.4) for 30 minutes followed by blocking and permeabilization for 1 h in PBS with 1% BSA and 0.1% Triton X-100 at room temperature. Caco-2 cells were then incubated at 4°C with anti-NFkB p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in a humidified chamber and incubated with a FITC-conjugated goat anti-rabbit antibody at room temperature and with Hoechst 33342 (10 µg/mL) for 5 min.
For all immunofluorescence studies, slides were mounted with Vectashield Mounting Medium with DAPI (Vector laboratories, Ltd, UK). Fluorescence images from multiple fields were obtained using a Nikon Eclipse e 80i microscopy. The images were analyzed using NiS Elements D imaging software (Nikon Instruments Inc., NY, USA).

DCFH-DA spectrofluorometry was used to measure ROS production in our model. DCFH-DA (20 µM) was added for 30 minutes at 37°C in the dark after stimulation. Intracellular ROS levels was measured in a fluorometer (Kontron Instruments, Japan). For DCF fluorescence imaging, Caco-2 cells were grown for 3 days on the cover glass, then fixed and permeabilized with paraformaldeyde 4% and Triton 0,2% for 30 minutes at 4°C. DCF-HA 20 µM was added for 30 minutes at 37°C in the dark. Fluorescence images from multiple fields were obtained using a Nikon Eclipse e 80i microscopy. The images were analyzed using NiS Elements D imaging software (Nikon Instruments Inc., NY, USA).

Embryo images were captured on a Nikon SMZ18 Stereo Microscope and processed with NIS-Elements D Imaging Software (Nikon), ImageJ, and Adobe Photoshop.

ROS were assayed by addition of 10 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA, Sigma-Aldrich, St Louis, MO, USA) for 30 min, and they were further washed with cold phosphate-buffered saline and observed under a Nikon eclipse CiS/Ci-L microscope and process with Nis elements D imaging software (Nikon Instruments, Amsterdam, The Netherlands). Quantification of ROS was done with Image J 1.48V (National Institutes of Health, Bethesda, Maryland, USA). The mean intensity of pixel was calculated for each image. Data for each biological replicate are the mean of seven fields per condition.

After microcomputed tomography analysis, the specimens were decalcified with 10% disodium ethylenediamine tetraacetate (pH 7.4) (Nacalai Tesque, Inc., Kyoto, Japan) at 4 °C for 6 weeks and were embedded in paraffin (Leica Biosystems, Nussloch, Germany) through standard dehydration and paraffin infiltration steps. The paraffin-embedded tissues were cut at 4-μm thickness using a rotary microtome (Leica Biosystems, Nussloch, Germany) parallel to the sagittal plane of the right hemimaxilla. Histomorphometric evaluations included the histological observations of stained tissue sections examined under DXm1200 light microscopy (Nikon, Kanagawa, Japan) using the NIS-Elements D Imaging Software (Version 2.30; Nikon, Kanagawa, Japan). The images were analyzed using ImageJ software (version 1.52; National Institutes of Health, Bethesda, MD, USA). The region of interest was determined to be a 330 μm × 409 μm region in the mesial root socket, which was considered representative of the extraction socket area. Analysis was performed after obtaining three randomized tissue sections for each sample with five random images at 200× magnification.