Cbl202

LAT1 IHC was performed on frozen and paraffin-embedded tumor sections. Frozen sections (10 μm) were dried, fixed in 4% paraformaldehyde, quenched in 50 mmol/L NH₄Cl, and permeabilized by 0.5% Triton X-100. FFPE sections were deparaffinized, rehydrated, and heat-induced antigen retrieval was performed using citrate buffer (pH 6.0). LAT1 was detected using mouse anti-mouse/human LAT1 (Santa Cruz Biotechnology, sc-374232) or rabbit anti-human LAT1 (Abcam, ab208776). Endothelial cells were detected using rat anti-mouse CD31 (BD Pharmingen, 550274) and mouse anti-human CD31 (Abcam, 187377) antibodies. MDA-MB-231-BR3 cancer cells were detected using anti-vimentin (Millipore, CBL202 or sc-6260, Santa Cruz Biotechnology) or with anti-human nuclei (Millipore, MAB1281) antibodies. Primary antibodies were detected using secondary antibodies labeled with fluorochromes Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 568 (Molecular Probes). Cell nuclei were detected with DAPI. Alexa Fluor 488 anti-mouse Fc-gamma subclass 2a specific (Jackson ImmunoResearch Laboratories, 115–545–206) was used as a secondary antibody for detection of human vimentin (Millipore, CBL202). Whole skulls were fixed in a combined fixation/decalcification solution (Cal-Ex II, Fisher Scientific, CS511–1D) for 72 hours, sectioned, and stained with hematoxylin and eosin.