Dmrb microscope

For light microscopy, a Leica (Wetzlar, Germany) DM RB microscope was used, equipped with an Olympus SP-350 digital camera. Image acquisitions were performed using OLYCAM/IAS software (ATZ, Bari, Italy). For confocal microscopy a Leica (Wetzlar, Germany) TCS-SP2 microscope was used, equipped with a laser Ar/Ar Krypton λ 488 (green, for the Y-chromosome), Gre/Neon λ 543 (red, for Cdx2), and He/Neon λ 633 (blue, for TOPRO). Images were collected and analyzed by Interactive LCS software (Leica, Wetzlar, Germany).

Postnatal mice were euthanized by CO₂ asphyxia and dissected for cornea, limb and tendon samples. Tissues (or embryos) were fixed in 4% paraformaldehyde/PBS for 48 h at 4 °C. Femur length of 2 month old mice (5 of each genotype) was evaluated using a fine digital caliper. Bone samples were decalcified at 4 °C in 10% EDTA/0.1 M Tris–HCl, pH 7.4 and processed for paraffin embedding using standard methods. Six μm thick paraffin sections were cut and stained with Alcian blue/nuclear fast red. Sections were mounted with microkitt and photographed with a Leica DMRB microscope equipped with an Olympus DP70 camera. Growth plate height measurements were derived from pictures of selected sections showing comparable histological architecture. The total growth plate height was averaged from measurements done at 5 different sites along the width of the bone on 5 individual mouse femurs of each genotype.

All methods for IHC and IF of mouse tumors have been described^{33 (link),62 (link)}. Briefly, tumors were excised at days 16–18 after tumor cell inoculation and snap-frozen in Tissue Freezing Medium (OCT; Jung) or fixed with 10% neutral buffered formalin (3 h) and paraffin embedded. Sections (5–20 μm) were prepared and used directly (frozen tissue) or deparaffinized, rehydrated, and subjected to heat-induced epitope retrieval in citrate buffer (0.01 M sodium citrate pH 6; 20 min). For immunostaining, the following rabbit antibodies were used: anti-CD31 (MEC13.3, BD Bioscience), -SOD3 (Cloud Clone), -VEC (LS-B2138, LSBio), -HIF-2α (PAI-32216, Thermo Fisher Scientific), and -3-NT (#06–284, Cell Signaling). Sections were incubated with appropriate fluorescently conjugated secondary antibodies (Alexa 488 or 546, Molecular Probes) or with peroxidase-labeled anti-rabbit IgG (Dako), followed by amino ethyl carbazol (AEC; Enzo) and hematoxylin counterstaining. Sections were mounted with 4,6-diamidino-2-phenylindole (DAPI)-containing Fluoromount-G (SouthernBiotech; fluorescence analyses) or Dako Faramount Aqueous Mounting Media (Dako; conventional IHC) and analyzed with an Olympus FluoView 1000 confocal microscope with a ×60 1.4 oil plan-Apo objective or with a Leica (DM RB) microscope equipped with an Olympus DP70 camera.

Immunohistochemistry and immunofluorescence were performed on 5-μm paraffin embedded tissue sections. For immunohistochemistry, enzymatic Avidin-Biotin Complex-diaminobenzidine staining (Vector Labs) was used with haematoxylin used for counterstaining nuclei according to standard protocols. Immunohistochemistry was performed on soft tissue sarcoma and normal arterial and skeletal muscle tissue array (US Biomax, Rockville, MD, USA #SO801a). The following primary antibodies and concentrations were used for immunohistochemistry: anti-BrdU 1:40 (Abcam #ab6326), anti-cleaved caspase-3 Asp 175 1:300 (Cell Signaling #9661) anti-Ki67 1:100 (Abcam #ab15580), anti-phospho-S6 (S235/236, Cell Signaling #4858) and anti-HIF-2α (ThermoPierce #PA1-16510). The following primary antibodies and concentrations were used for immunofluorescence: anti-CD31 1:50 (Abcam #ab28364), anti-pimonidazole FITC 1:100 (Hypoxyproble HP2). Mounting media with DAPI (Life Technologies #P36935) was applied last. Sections were imaged using a Leica DMRB microscope and an Olympus DP72 camera.

On day 21 of culture, cells were fixed with either 70% ethanol for 60 min at − 20 °C for Alizarin Red staining or with 4% formalin for 60 min at room temperature for Oil Red or Alcian blue staining. Alizarin Red, Oil Red, and Alcian blue staining were performed at room temperature according to manufacturers' instructions. Briefly, Alizarin Red staining was performed for 10 min, followed by washes with distilled water and PBS; Oil Red staining was performed for 15 min; and Alcian blue staining for 120 min. Pictures were taken with LEICA DMRB microscope equipped with an Olympus DP70 digital camera, 10 ×/0.30 PL FLUOTAR objective or 40 ×/0.70 PL FLUOTAR objective, and the DP controller software.

Estimates of the number of Fos-positive cells in each region of interest were made using an automated cell counting procedure. Wherever possible, cell counting occurred without knowledge of the group assignments. (This blind procedure was not always possible for those sections that also contained lesioned areas.) Images were viewed on a Leica DMRB microscope and photographed using an Olympus DP70 camera. The programme cellSens (Olympus) counted the number of cells stained above a threshold of greyscale intensity that was above background level.
These counting procedures are not stereological and therefore do not provide absolute cell numbers, but rather provide a relative measure of numbers of IEG-positive cells, with various caveats (Coggeshall and Lekan, 1996 (link)). For all brain areas analysed, counts were taken from either three or four sections per hemisphere, depending on the brain area. The number of sections from a given area was the same for all animals. The cells counts for a given area were then averaged to produce a mean count.

Only mature (late phase) cells (Mandyam et al., 2008 (link)) were analyzed (Fig. 1B); the inclusion criteria were as described above. Plane images were captured under bright-field microscopy with a Leica Microsystems DMRB microscope equipped with a 20× (NA 0.50) objective, a CCD camera (Olympus, DP73), and Cell Sense Dimensions software 1.16 (Olympus). Images were converted to 8-bit grayscale and inverted before Sholl analyses (to a maximum distance of 300 μm from the soma). Dendritic arbors were traced from a total of 158 cells: 88 cells for MTT-lesioned cases and 70 cells from surgical controls with 5–16 cells analyzed per case. The mean number of intersections per animal was then calculated.