Tissue pretreatment buffer solution

For hepatic LHBS staining, paraffin-embedded tissues were pre-warmed at 65°C for 15 min and treated with xylene for paraffin removal. Antigen retrieval was performed by boiling the samples in a tissue pretreatment buffer solution (Invitrogen) for 30 min. The slides were then incubated with mouse anti-preS1 for 1 hr and assessed using the EnVision+ System-HRP kit (DAKO, Carpintena, CA, USA). For double immunofluorescence staining, the cells were fixed and stained with the indicated primary antibodies for 1 hr, followed by secondary antibodies conjugated with Alexa-488, Alexa-594, or Alexa-647 (Molecular Probes). The cells were mounted with mounting medium that contained DAPI. The images were acquired with a Zeiss LSM780 confocal microscope or an automated Leica DMI6000 inverted microscope equipped with an HCX PL FL 20x/NA0.4 objective and an EMCCD camera (Andor Luca R, Belfast, UK) as indicated. The following primary antibodies were used in this study: mouse anti-preS1 (a gift from Ningshao Xia, Xiamen University, China), rabbit anti-SNAP tag (P9310S, New England Biolabs, MA, USA), rabbit anti-STIM1 (PA5-23623, Thermo Scientific, IL, USA), and mouse anti-γ-tubulin (sc-17787, Santa Cruz, Dallas, Texas, USA).

Paraffin-embedded samples were pre-warmed at 65°C for 15 min, followed by treatment with xylene to remove the paraffin. The samples were rehydrated by sequential incubation in 100%, 95%, and 70% ethanol and then in water. Antigen retrieval was performed by boiling the samples in tissue pretreatment buffer solution (Invitrogen) for 30 min. The slides were then incubated with mouse anti-Sgo1 antibody (Abnova) for 1 hour and detected using the EnVision+ System-HRP kit (DAKO). The samples were counterstained with hematoxylin (DAKO) and imaged under a Zeiss LSM780 laser confocal microscope or a Leica DMI6000 microscope.