Dfc300fx camera

Spinal slices were used for the detection and quantification of GFAP or Iba-1 immunofluorescence. After preincubation for 1 h with Tris-buffered saline with 0.1% Triton X-100 (pH 7.5), sections were sequentially incubated overnight at 4 °C with the following polyclonal antibodies: (i) anti-Iba-1 (Wako Chemicals, Richmond, VI, USA) used at 1:500; or (ii) anti-GFAP (Dako Cytomation, Glostrup, Denmark) used at 1:200, followed by washing in Tris-buffered saline and a new incubation (at 37 °C for 2 h) with an anti-rabbit secondary antibody conjugated with Alexa 488 (Invitrogen, Carlsbad, CA, USA). A DMRB microscope and a DFC300Fx camera (Leica, Wetzlar, Germany) were used for slide observation and photography. The mean density of immunolabelling was measured in the selected areas. Again, all data were transformed to the percentage over the mean obtained in the wild-type group for each parameter.

For immunofluorescence see procedure described Perez-Asensio et al. (2013) (link). The list and dilution of primary antibodies was: pSTAT3_ser727 (1:500), pERK1/2 (1:500), BIII-Tubulin (1:1000), DCX (1:1000), Nestin (1:200), Ki67 (Leica Microsystems, Wetzlar, Germany 1:1000), BrdU (AbCam, 1:400), Olig2 (Millipore, 1:400), GFAP (DAKO, 1:2000). Staining on cell cultures was visualized with a Leica CTR400-DMI400B inverted microscope or Leica DM5500Q confocal microscope. Photographs were taken by a DFC300FX camera from Leica. Quantification was carried out using Leica Application Suite (LAS-Leica) or ImageJ (NIH) software. The number of positive cells for each experimental assay was expressed as percentage of the total number, and/or referred as percentage of the control.

Wound-healing assays were performed by creating identical wound areas into the cell monolayer using Ibidi culture-inserts (Ibidi GmbH, Cat. no. 80209, Munich, Germany). U2OS cells were seeded in complete culture medium at a density of 3 × 10⁴ cells on each side of the Ibidi culture-insert, into a 24-well plate. After attachment, cells were treated for 24 h inhibitors (or vehicle in the controls); then the culture-insert was detached in order to form a cell-free gap into the cell monolayer. Each well was rinsed once with phosphate-buffered saline (PBS) to remove cell debris and immediately refilled with fresh medium, with new addition of the inhibitors (samples with “maintained” treatment) or without (sample with “removed” treatment). Cells were allowed to migrate for further 48 h. The wound images were captured at time zero (t = 0 h), t = 24 h, and t = 48 h using a Leica DMI4000 automated inverted microscope equipped with a Leica DFC300 FX camera.

Slices were used for Nissl staining using Cresyl Violet, as previously described (Alvarez et al., 2008 (link)). A Leica DMRB microscope (Leica, Wetzlar, Germany) and a DFC300FX camera (Leica) were used for the observation and photography of the slides, respectively. For counting the number of Nissl-stained large motor neurons in the anterior horn, high-resolution photomicrographs were taken with the 20× objective under the same conditions of light, brightness and contrast. Four images coming from at least three sections per animal were analysed. The final value is the mean for all animals included in each experimental group.

‘Clean’ injury referred to an injury performed with a needle that has been previously sterilized. A low level of bacterial contamination is still possible since the surface of the insect was not sterilized. For adults, two different intensities of physical wounding were used (adapted from [13] (link)). In strong wounding the thorax of the fly was completely penetrated using a sterile needle of ∼50 µm diameter. For survival to clean injury and for imaging of the melanization reaction upon pricking, the thorax of the animal was pricked (as described in infection experiments) using a sterile needle (diameter: ∼5 µm). Pictures were taken three hours post-pricking. Third instar larvae were pricked dorsally using a sterile needle (diameter: ∼5 µm). Pictures of melanized larvae were taken 30 minutes post-injury. For the melanization time-course of larvae (Fig. 3B), the presence of a blackening spot was recorded after pricking, every 5 minutes until 30 minutes. Pictures were captured with a Leica DFC300FX camera and Leica Application Suite.

U2OS cells were plated at 50% confluence, allowed to adhere to the plate for 16 h, and then treated with inhibitors or vehicle for 24 h. After treatment, cells were washed, detached, counted, and seeded at 1000 cells/well in 200 μL of culture medium, in a 96-well plate with round bottom wells precoated with 50 μL of 1% agar in culture medium [17 (link)]. Images were taken from each well 24 h and 96 h later, by means of a Leica DMI4000 automated inverted microscope equipped with a Leica DFC300 FX camera.

Wound-healing assays were performed by creating identical wound areas into the cell monolayer using Ibidi culture-inserts (Ibidi GmbH, Cat. no. 80209). Cells were seeded in triplicate in complete culture medium on each side of the Ibidi culture-insert, into a 24-well plate, at proper cell densities (1.5 × 10⁶ SK-N-BE cells/mL and 5.5 × 10⁴ U2OS cells/mL). After 24 h, cells reached confluence, and the culture-inserts were removed in order to form a cell-free gap into the cell monolayer. Each well was washed once with PBS to remove cell debris and immediately refilled with fresh D-MEM medium supplemented with 5% (v/v) FBS and 2mM L-glutamine. The wound images were captured at this time (t = 0) and at the indicated time points (24–48 h for SK-N-BE, 2–4–8 h for U2OS) using a Leica DMI4000 automated inverted microscope equipped with a Leica DFC300 FX camera (2.5× objective, magnification-changer 1.6). Images of in vitro scratch wound healing assays were analyzed with a plugin of ImageJ software (1.52t version) adapted from [28 (link)] and relative cell migration of each cell line was measured.