Dp50 digital camera

Disruption of the Δψm was measured using a MitoLight Mitochondrial Apoptosis Detection kit (Chemicon, Temecula, CA, USA) according to the manufacturer’s instructions. MitoLight solution was added to the cultured cells and incubated at 37°C in a 5% CO₂ incubator for 15 min. Cells were then washed in PBS to remove excess MitoLight and examined under a fluorescence microscope (BX60, Olympus, Tokyo, Japan). Images were captured using a DP50 digital camera (Olympus). MitoLight aggregates in the mitochondria of healthy cells and fluoresces red. MitoLight cannot accumulate in the mitochondria of apoptotic cells; it remains as monomers in the cytoplasm and fluoresces green.

On the 7th day post‐operation, rats were deeply anaesthetized with sodium pentobarbital (50 mg/kg, ip) and intracardially perfused with saline. Next, 4.0% paraformaldehydein 0.1 mol/L PBS (pH = 7.4, Sigma) was used for perfusion. Spinal cord segments L4‐L6 were extracted and fixed at 4°C for 12 hours in the same fixative, and then transferred to PBS containing sucrose (15%‐20%). On the next day, the segments were sliced continuously at a thickness of 30 μm. Free floating sections were subsequently stained using the standard avidin‐peroxidase complex (ABC) method. The sections were incubated overnight in primary rabbit monoclonal antibody to CD11b (ab133357, 1:250, Abcam) in 0.1 mol/L PBS containing 5% normal goat serum and re‐probed with diluted biotinylated goat anti‐rabbit IgG (1:200). The product was visualized with 0.03% hydrogen peroxide and 0.05% 3, 3′‐diaminobenzidine (DAB) solution as chromogen. The sections were then fixed on glass slides, dehydrated by gradient ethanol, dehydrated with xylene, permeabilized and mounted. The sections were observed under a brightfield Olympus BX51/BX52 microscope. Images were obtained using an Olympus DP50 digital camera and processed using the Olympus DP Image software (version 3.1).

Whole-mount LRT specimens were photographed on a Leica M165FC microscope equipped with a DFC310FC digital camera and Application Suite software (ver. 3.3.0; Leica Camera AG, Solms, Germany). Histological sections were photographed on an Olympus AX80 microscope equipped with a DP50 digital camera and Studio Lite software (ver. 1.0; Olympus corporation). Some images were merged and trimmed using Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA, USA).

On the day of dissection, animals were deeply anaesthetized and perfused via the aorta with cold phosphate buffered saline (PBS, 0.1 M, pH 7.4), followed by 4% paraformaldehyde (PF) in phosphate buffer (PB, 0.1 M, pH 7.4). Dissected tissues (lumbar spinal cord, and L4, 5 DRGs) were postfixed (1–1.5 hours) and cryoprotected in 20% sucrose. Control and experimental tissues were embedded in the same cryomolds, covered in OCT and frozen in cooled isopentane prior to storing at −80°C until processing.
Sections were analysed and photographed with an Olympus BX-60 microscope connected to an Olympus DP-50 digital camera. Brightness and contrast were adjusted with the software Adobe Photoshop 5.0.

The adult flies were weighed using an Analytical Semi-Micro Balance (A&D Company, Tokyo, Japan). To measure wing size, the right wings of the adult flies were torn off by using forceps and mounted onto a microscopic slide using a drop of Fly Line Dressing (TIEMCO, Tokyo, Japan), a silicone grease with very low surface tension (Tsuda et al., 2010 (link)). The wings were photographed using a MZ APO stereomicroscope (Leica, Wetzlar, Germany) equipped with a DP50 digital camera (Olympus, Tokyo, Japan) at a constant magnification. The areas of the wings were measured by using ImageJ software (NIH).