*4.6. Immunohistochemistry*

Cell-gel constructs were fixed with formaldehyde and incubated for 2 h at room temperature with blocking buffer. Immunolocalization of MMP-13 was performed by using rabbit polyclonal anti-human MMP-13 (1:1000) (ab84594; Abcam, Cambridge, MA, USA) overnight at 4 ◦C. The secondary antibody alexa-555 goa<sup>t</sup> anti-rabbit (1:2000 dilution) (A31630; Invitrogen) was incubated for 2 h at room temperature. Negative control staining was performed with Dako rabbit negative control (Dako, Glostrup, Denmark). Nuclei were stained with <sup>4</sup>,6-diamidino-2-phenylindole (DAPI).

Six μm optical sections were made of the gels with an Axio ZoomV16 microscope (Zeiss, Munich, Germany). The micrographs were then superimposed, and the number of positive cells was counted. With this technique, we were able to scan through 200 μm gel and count the MMP-13-positive cells in these areas.
