Dxm 1200 camera

At 9 DAT and 12 DAT, the fruits were sliced along the equator. After removing the seeds and pulp, the pericarps were cut at the equatorial level and further divided into approximately 5 mm wide pieces before being immersed in formaldehyde, alcohol, acetic acid (FAA) fixative. These pericarp pieces were then embedded in paraffin and stained with Safranin-O and Fast Green, following a previously described method (Takacs et al., 2012 (link)). The slides were observed and photographed using a Leica DFC7000 T microscope (Wetzlar, Germany) equipped with a Nikon DXM 1200 camera. To estimate the pericarp thickness and the number of cell layers, six samples of the pericarp without vascular bundles were examined.

Fluorescence images were acquired by confocal microscopy (Zeiss LSM 710). In double-labeling experiments, images were sequentially acquired. All images were further processed in Adobe Photoshop 6.0, with only contrast and brightness adjustments. Photomicrographs labeling were taken on a Nikon Eclipse E600 light microscope using a DXM 1200 Camera equipped with ACT-1 software. The microscope was set at a specific illumination level, as was the camera exposure time. c-Fos-positive nuclei were counted using NIH ImageJ software. Labeling and counting were performed on every fourth coronal section throughout the rostro ¬¬–¬caudal axis from the brainstem to the hypothalamus.

Mice were euthanized by cervical dislocation. To obtain anterior and posterior parts of the brain, mouse brains were carefully removed and cut in coronal plane between bregma 0 and −2.12 mm. Right and left hemispheres were then separated along the sagittal plane. Then, the brains were immediately fixed in Bouin’s solution for 24 h and later embedded in paraffin blocks. Serial sections of mouse brains (5 μm) were deparaffinized and rehydrated. Endogenous peroxidase activity in the tissue was reduced by treatment with 3% H₂O₂ in 100% methanol. Then, the sections were incubated for 2 h in blocking reagent (4% normal goat serum in 0.1 M PBST pH 7.4). The sections were incubated for 2 h with polyclonal antibody against tyrosine hydroxylase (1:1,000). Subsequently, the sections were washed and then incubated with secondary antibody, HRP-conjugated goat anti-rabbit IgG (1:4,000) for 1 h. Peroxidase activity was visualized by reacting with DAB in 0.05 M tris-buffered saline (pH 7.6). The stained tissues were observed and photographed under Nikon E600 microscope equipped with Nikon digital DXM1200 camera, using an ACT-1 software package. Tyrosine hydroxylase (TH) intensity in the SNpc and striatum was measured in TH-positive cells and fibers, and quantified with image J version 1.51k.

Longitudinal sections were photographed with objectives of x1 magnification with a Nikon E600 light microscope and a Nikon DXM 1200 camera (Nikon Instruments, Melville, NY, USA) connected to a PC. All slides were photographed under the same conditions for exposure time, light intensity, and camera gain. Analyses were performed by one evaluator (AmT) using NIS-elements imaging software (NIS-elements Basic Research, Nikon, Tokyo, Japan). White balance adjustments, calibrations, and transformations of the photographs into binary images were performed for each captured image. The measurements were performed on an area restricted by the defect, the ulnar bone and the outer border of the graft material (Fig 7). The total area and the area filled by graft material and bone (binary area) were measured. The fraction of binary area:total area was calculated as percentage.

Pum2^–/– and wildtype littermates were perfused with 4% paraformaldehyde (PFA) and whole brains were dissected. The brains were in same fixation overnight at room temperature, transferred to 20% sucrose/PBS buffer for 24 hours, then embedded in Tissue-Tek O.C.T. (Electron Microscopy Sciences, Hatfield, PA, USA). Brains were cut in coronal section at 40 μm and mounted on slides. Sections were fixed in 2% PFA/PIPES buffer for 10 min on ice, and rinsed with Rinse Buffer (2 mM MgCl₂/PBS) for 10 min on ice, then incubated in X-gal solution at 37° C overnight. Sections were rinsed with Rinse Buffer, 2% PFA/PIPES buffer, PBS + 2 mM MgCl_2, (each for 5 min) then counterstained with Neutral Red and dehydrated with ethanol and mounted with Permount. Slides were examined on a Leica MRT compound microscope (Leica Camera AG, Solms, Germany), and images were captured with the Nikon DXM1200 camera and native ACT-1 image software (Nikon Corporation, Tokyo, Japan).