Axiocam icc1 camera

Skin substitute biopsies were fixed in HistoChoice solution (AMRESCO, Inc., Solon, OH, US) and embedded in paraffin. Five µm thick sections were cut and were stained with Masson’s trichrome staining. Two substitutes for each of the 3 donors were analyzed (n = 6). Skin sections were observed using a Zeiss microscope equipped with an AxioCam ICc1 camera (Carl Zeiss Meditec, AG, Oberkochen, Germany). The thickness of the dermis and epidermis was measured on the stained sections using ImageJ software (National Institutes of Health, Bethesda, MD, US; http://imagej.nih.gov/ij). A total of 10 measurements on three sections of each biopsy were made.

1×10⁵ cells/cm² of B16V cell lines were seeded in 12-well plates in RPMI medium and starved overnight. An artificial wound was generated and cells were incubated with serum-free RPMI medium (control) or RPMI supplemented with 10 ng/ml Fgf2 for 20h at 37°C. Images were captured at time points 0 and 20h, using a Zeiss Axiovert 100 microscope with AxioCam ICc1 camera. Cell migration was evaluated as described [5 (link)]. For each well 2–4 pictures were acquired (n = 3 independent experiments).
Primary C57BL/6 skin fibroblasts were cultured for 10 days in 35 mm petri dishes with 1 mM L-ascorbate-2-phosphate (Sigma) to obtain a 3D ECM [37 (link)]. Confluent B16V cell lines were detached from the culture dish with trypsin/EDTA, and B16V cell suspension in serum-free RPMI1640 was added to the 10 day old and 24h serum-starved C57BL/6 fibroblast cultures. Migration of cells was monitored, evaluated and calculated as described before [38 (link)].

Wild-type and transgenic P. yoelii sporozoites (PY17X_1354300::GFP, PyUIS12::GFP) were subjected to live fluorescence assays and/or an indirect immunofluorescence assay (IFA)^{72 (link)} to characterize the extent of translational repression of these candidates. For live fluorescence microscopy of PyUIS12::GFP, freshly produced salivary gland sporozoites placed on glass slides in VectaShield, overlaid with a cover glass slip, and visualized by fluorescent microscopy using a Zeiss Axioscope A1 with 8-bit AxioCam ICc1 camera and Zen imagine software from the manufacturer. Alternatively, fresh salivary gland sporozoites were fixed in 10% v/v formalin for 10 min, and then air dried to a well on a glass slide defined by a hydrophobic coating. Sporozoites were treated for IFA using either rabbit polyclonal anti-PyUIS4 (antigen consisting of AA80-224, diluted 1:1000), produced by Pocono Rabbit Farm and Laboratory, Canadensis, PA) or rabbit polyclonal anti-GFPmut2 (diluted 1:1000) as primary antibodies and anti-rabbit IgG antibodies conjugated to Alexa Fluor 488 as a secondary antibody (diluted 1:500).

A linear scratch on confluent MC3T3-E1 cells was performed by a sterile pipette. We removed the cellular debris by washing with PBS, and then we incubated the cells in DMEM 1% FBS alone or in the presence of various treatments for 24 h (see [41 (link)] for reference). We took the photographs immediately after the scratch (t0) and at various times after treatment (t18, t24, and t48) using an Olympus U-CMAD3 phase-contrast microscope equipped with a Zeiss Axiocam Icc1 camera at a 4× magnification. We analyzed the images using ImageJ software (NIH, Bethesda, MD, USA) as previously reported [42 (link)]. The covered area percentage was calculated for each experimental group by measuring the wound size at different times from treatment compared with the initial (t0) wound size considered as 100%.

Cross sections (5 μm) of paraffin-embedded tissues were also processed for Picrosirius red staining (Direct Red 80, Sigma) to allow collagen content quantification. Images were taken using the Zeiss Axio Imager M2 microscope with an AxioCam ICc1 camera (Zeiss Canada) with a polarized light. Multiple images were taken per sample, and reconstitution of the entire sample was achieved using the Photoshop CS4 software (Adobe) and the repositioning photomerge function.
The histological collagen fiber content was quantified on the Picrosirius red-stained tissues from NONcNZO10/LtJ mice (n = 7 wounds per group) using the ImageJ software (National Institutes of Health) and the Color Inspector 3D plugin. The surface analyzed corresponded to the entire granulation tissue area. A graph was generated representing the color composition of the delimited area. A threshold was used to isolate the three main colors (red, yellow, green) seen in Picrosirius stained samples. Red represents mature collagen type I fibers, while green/yellow represents immature collagen type III fibers [47 (link)]. The relative proportion of each of these three colors was quantified by measuring the ratio of the pixel count of each color on the total pixel count for each image.

To observe the morphological aspect of c-KIT+ and c-KIT- cells, we spread a drop from each cell suspensions over the glass-slide. Then the slides were allowed to air dry for 30 minutes and fixed for 10 minutes with 4% formaldehyde. At the end the slides were stained with Papanicolaou stain and reviewed by a senior cytopathologist under the inverted microscope CARL ZEISS Axio Observer Z1FLMot, who evaluated the main characteristics of malignancy: nuclear pleomorphism and hyperchromasia, ratio nucleus/cytoplasm, coarse chromatin pattern, multinucleation, pleomorphism of nucleoli and presence of multiple nucleoli. Images were taken with CARL ZEISS AXIOCAM Icc1 camera.