Sc 8002 af594

Frozen sections (10 μm) were dried, fixed in 4% paraformaldehyde, quenched in 50 mM NH4Cl and permeabilized by 0.5% Triton X-100. MDA-MB-231-BR3 cancer cells were detected using antibodies against human vimentin (Millipore, CBL202) followed by a secondary Alexa Fluor 488 anti-mouse Fc-gamma subclass 2a specific antibody (Jackson ImmunoResearch Labs, 115–545-206). The following secondary antibodies were used in other applications—Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 568 (Molecular Probes). MCF7 cells were detected by anti-Pan-cytokeratin antibody (Novus Biologicals, NBP2-33200) or mouse monoclonal anti-human estrogen receptor alpha antibody (Santa Cruz Biotechnology, sc-8002-AF594). Endothelial cells were detected using rat anti-mouse CD31 (BD Pharmingen, 550274) or rat anti-mouse PV1 (BioRad, MCA2539T) antibodies. Cell nuclei were detected with DAPI. Whole skulls were fixed for 72 h using a combined fixation and decalcification protocol (Cal-Ex II, Fisher Scientific, CS511-1D), sectioned and stained with hematoxylin and eosin.

Frozen sections (10 μm) were dried, fixed in 4% paraformaldehyde, quenched in 50 mM NH4Cl and permeabilized by 0.5% Triton X-100. MDA-MB-231-BR3 cancer cells were detected using antibodies against human vimentin (Millipore, CBL202) followed by a secondary Alexa Fluor 488 anti-mouse Fc-gamma subclass 2a specific antibody (Jackson ImmunoResearch Labs, 115–545-206). The following secondary antibodies were used in other applications - Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 568 (Molecular Probes). MCF7 cells were detected by anti-Pan-cytokeratin antibody (Novus Biologicals, NBP2–33200) or mouse monoclonal anti-human estrogen receptor alpha antibody (Santa Cruz Biotechnology, sc-8002-AF594). Endothelial cells were detected using rat anti-mouse CD31 (BD Pharmingen, 550274) or rat anti-mouse PV1 (BioRad, MCA2539T) antibodies. Cell nuclei were detected with DAPI. Whole skulls were fixed for 72 hours using a combined fixation and decalcification protocol (Cal-Ex II, Fisher Scientific, CS511–1D), sectioned and stained with hematoxylin and eosin.