Mouse anti melana

Cells were fixed in 4% paraformaldehyde and incubated in PBS-0.05% Triton X-100 (PBST) containing 1% normal goat serum (NGS; Invitrogen) for 30 minutes to block non-specific antibody binding sites. The cells were then incubated with mouse monoclonal antibodies against vinculin (Sigma-Aldrich) at 4 °C for 12 h. This step was followed by incubation with Alexa Fluor 546 conjugated anti mouse IgG (Molecular Probes, Invitrogen), Hoechst (invitrogen), and Alexa Fluor 488 conjugated phalloidin (Molecular Probes, Invitrogen). Finally, the cells were washed with PBST and mounted using the Prolong Gold antifade reagent (Molecular Probes, Invitrogen). Fluorescent images were observed using a fluorescence microscope, and processed using the Adobe Photoshop 10.0 software.
To identify the cancer cells, the following antibodies were applied as cancer cell markers; mouse anti MelanA (Santa Cruz) for B16F10, mouse anti CathepsinD (Abcam) for MDA-MB-231, and mouse anti PSA (Abcam) for MDA-PCa-2b. Intercellular adhesion was visualized using the following antibodies: rabbit-anti Connexin 43 (Cell Signaling) and rabbit anti-Cadherin 11 (Cell Signaling).

The cultured femurs were fixed in neutral buffered formaldehyde for 24 h, followed by decalcification using 0.5 M EDTA-2Na solution (pH 7.4) for 7 d. Specimens were dehydrated through a graded series of ethanol, embedded into paraffin, and transversely cut into 5 μm-thick sections. Deparaffinized sections were incubated with normal goat serum (Thermo Fisher Scientific, Waltham, MA, USA) to block non-specific antibody binding sites. The specimens were then incubated with rabbit anti MMP1 (Abcam, Cambridge, UK), mouse anti melanA (Santa Cruz, Dallas, TX, USA), mouse anti ALP (Novus Biologicals, USA, Centennial, CO, USA), and rabbit anti Cx43 (Cell Signaling, Danvers, MA, USA) antibodies. The secondary antibodies used were as follows: Alexa Fluor 546-conjugated anti-rabbit IgG (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA), and Alexa Fluor 488-conjugated anti-mouse IgG (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with DAPI (Thermo Fisher Scientific, Waltham, MA, USA).