Immunofluorescence Analysis of Mammary Gland Tissues

Immunofluorescence analyses were performed using the reported methods [26 (link), 27 (link)]. The mammary gland tissues were fixed, dehydrated, and embedded in paraffin. Sections of the tissues (3 μm thick) were then air-dried on MAS-coated slides. After deparaffinization, antigen retrieval was performed by autoclaving the sections in a citric acid buffer (pH 6.0) for 20 min at 121 °C. Cultured GMECs on collagen gel isolated from the insert were fixed with methanol for 10 min at − 20 °C and then with 1% paraformaldehyde in PBS for 10 min at 4 °C. Subsequently, the tissue sections and cells were washed with PBS for 10 min, after which they were incubated in PBS-T (PBS containing 0.05% Tween-20) containing 5% bovine serum albumin (MP Biomedicals) for 1.5 h at room temperature. The sections and cells were then incubated overnight at 4 °C with rabbit polyclonal antibodies against L-amino acid transporter (LAT)-1 (#ab208776; Abcam, Cambridge, UK, 1:100), LAT3 (#ab254719; Abcam, 1:100), and claudin 3 (#34-1700, Thermo Fisher Scientific, 1:200) or with mouse monoclonal antibodies against occludin (#sc-133,256; Santa Cruz Biotechnology, 1:200) diluted in PBS-T containing 2.5% bovine serum albumin. To evaluate immunofluorescence, the sections and cells were incubated with secondary antibodies (Alexa Fluor 488–conjugated goat anti-rabbit, #A32731, 1:400; Alexa Fluor 555–conjugated goat anti-mouse, #A32727, 1:400; both Thermo Fisher Scientific) diluted with PBS-T containing 2.5% bovine serum albumin for 1 h at room temperature. Immunofluorescence images were obtained using a fluorescence microscope (BZ-9000) and processed using analysis software (Keyence, Osaka, Japan).