Goat anti chicken igy

For in situ hybridization assay, paraffin sections (10 µm thickness) were pretreated with proteinase K and hybridized with appropriate digoxigenin-labeled probes, as described previously (St Amand et al. 2000 (link)). Transcripts were detected by color reaction with BM purple (Roche) and counterstaining with eosin. Immunofluorescence was performed on sections of 6 µm in thickness by using anti-Ki67 antibody (Cell Signaling Technologies, #9129) and MF-20 antibody (Developmental Studies Hybridoma Bank), respectively, following the manufacturers’ instructions. After incubation with secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG or goat anti-chicken IgY, Invitrogen), the samples were counterstained with 4,6-diamidino-2-phenylindole (DAPI), and visualized under a fluorescence microscope. Ki-67-positive cells at the condyle and glenoid fossa region were counted and presented as the percentage of total cells within the defined areas. Student’s t-test was applied to determine the significance of difference between wild-type controls and mutants (n = 3 for each genotype). Apoptosis analysis (Tunel) of TMJ was conducted by using the In Situ Cell Death Detection Kit (Roche) and alkaline phosphatase staining (n = 3 for each genotype).