Spot rt digital camera

Light microscopy was performed using standard methods. Freshly removed heads were partially dissected to remove the mouthparts, frons and the cuticle from the back of the head. The dissected head capsule was kept in primary fixative (2.5% glutaraldehyde and 4% paraformaldehyde in 1M phosphate buffer saline, PBS) for 3 h. After fixation, the samples were post-fixed in 1% phosphate buffered osmium tetroxide to enhance the contrast, and through an ethanol series (50–100%) for dehydration. The samples were then embedded in hard Epoxy resin (Epon® 812) and carefully oriented at different angles for sectioning. Semi-thin sections of 1 μm were cut using either a glass or diamond knife on a Reichert-Jung ultramicrotome. Sections were post-stained with toluidine blue and imaged on a Zeiss Axioskop (light microscope) with a SPOT RT digital camera.

Immunohistochemical staining was performed to analyze beta-catenin expression and presence of administered humanized Scl-Ab. The phosphate buffered formalin was used to fixe femora that were then decalcified by 5% formic acid for two weeks, embedded in paraffin and sectioned into five-micrometer thickness. After removal of paraffin and rehydration, sections were quenched with 0.5% hydrogen peroxide for 20 min, and treated with 10 mM citric acid for 10 min at 65 °C for antigen retrieval. Blocking was performed using 5%BSA in 1x PBS for 1 hr at room temperature. The sections were incubated with either rabbit monoclonal anti-beta-cetenin antibody (1:200, Abcam, Cambridge, MA, US) or rabbit anti-Human IgG antibody (1:200, Abcam), in a humid chamber for overnight at 4 °C. These sections were then incubated with horseradish peroxidase conjugated anti-rabbit IgG antibody (Abcam) at room temperature for 30 min and developed using DAB (Thermo Scientific, Fremont, CA, US). These sections were evaluated under light microscope (Zeiss Aixoplan with Spot RT digital camera, Zeiss, German).