Pd70 ix2 ucb microscope

Femur explants were fixed in 4% paraformaldehyde and embedded in paraffin. Serial 5 μm sections were stained with hematoxylin–eosin-safran reagent using standard protocols. For immunohistochemical assessment, sections were labeled with the following antibodies and a Dako Envision Kit: anti-Col X (BIOCYC, N.2031501005; 1:50 dilution), anti-Sox9 (polyclonal antibody, Santa Cruz Biotechnology Inc., catalog D0609; dilution 1:75), anti-phosphorylated ERK1/2 (Thr180/Tyr182) (Cell Signaling Technology, #4370; 1:100 dilution), anti-phosphorylated p38 (Abcam, Ab4822; 1:200 dilution), and anti-Ki-67 (Abcam, Ab16667; 1:3 000 dilution). Images were captured with an Olympus PD70-IX2-UCB microscope and quantified using cellSens software.

The femurs and cranial base from Osx-cre, Osx-Fgfr3 and Col1-Fgfr3 mice and their control littermates were fixed in 4% paraformaldehyde at 4°C for 24 h, washed in PBS, decalcified in 0.5 M EDTA (pH 8.0) for a week or a month (depending on mouse age), dehydrated in graded ethanol solutions, cleared in xylene and embedded in paraffin. Five-micrometer sections were cut, stained with Safranin O and immunohistochemically stained using standard protocols. Antibodies against collagen type X (1:50; BIOCYC, Luckenwalde, Germany), pERK1/2 (1:500; Cell Signaling Technology, USA) and Ki67 (also known as Mki67; 1:3000; Abcam, Cambridge, MA, USA) and the Dako Envision kit (Dako North America, CA, USA) were used. Images were acquired with a PD70-IX2-UCB microscope (Olympus, Tokyo, Japan) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Mean areas of individual HCs and number of HCs were measured from collagen X-labeled sections, within multiple 8500 µm² regions of interest on sections immunostained for collagen type X.

After a 6-day culture period, fetal femur (E16.5) explants were fixed in 4% paraformaldehyde, decalcified with EDTA (0.4M), and embedded in paraffin. Serial 5 μm sections were stained with hematoxylin‑eosin‑safran (HES) reagent, using standard protocols. For immunohistochemical assessment, sections were labeled with the following antibodies and a Dako Envision Kit: anti-COLX (BIOCYC, catalog N.2031501005; 1:50 dilution), and anti–phosphorylated ERK1‑2 (Thr180/Tyr182) (Cell Signaling Technology, catalog 4370; 1:100 dilution). Images were captured with an Olympus PD70-IX2-UCB microscope and quantified using cellSens software.
Mean areas of individual hypertrophic chondrocytes were measured from COLX-labeled sections, within a 166 μm wide × 76 μm high box positioned 50 μm from mineralization front (Supplemental Figure 6). The measurements were made manually using Fiji software and the freehand selection tool. For analysis of the effect of the drug treatments on the area occupied by proliferative chondrocytes, these cells were identified by their round or columnar shape, as seen with HES staining, and by the absence of COLX labeling. We measured the total area occupied by chondrocytes within the whole growth plate and the area occupied by COLX⁺ chondrocytes. The area for proliferating chondrocytes was calculated by subtracting the COLX⁺ area from the whole growth plate area.