Hoechst 33342

At 24 h posttransfection, the cells were fixed with 4% paraformaldehyde for 30 min. After the cells were washed three times with PBS, they were incubated in blocking buffer (3% bovine serum albumin, 0.1% Triton X-100 in PBS) at room temperature for 30 min. For the staining of myc-tagged NiV V, UBXN1 and HA-tagged UBXN1, anti-myc rabbit polyclonal antibody (Sigma), anti-myc mouse monoclonal antibody (Clontech), anti-UBXN1 rabbit polyclonal antibody (Millipore) and anti-HA mouse monoclonal antibody (Sigma) were incubated with the cell in blocking buffer at 4 °C for over night. For control staining, rabbit serum was used. After the cells were washed three times with wash buffer (0.05% Tween 20 in PBS), they were incubated with Alexa-Fluor-488-conjugated goat anti-rabbit antibody (Invitrogen), Alexa-Fluor-568-conjugated goat anti-mouse antibody (Invitrogen), and Hoechst 33342 (Cambrex) in blocking buffer at room temperature for 1 h. After the cells were washed three times, their immunofluorescence was observed with an IX70 laser confocal microscope and the FluoView FV1000 system (Olympus).

Tumour tissues were fixed in 4% PFA overnight at 4 °C, embedded in Tissue-Tek OCT compound (Sakura Finetechnical Co., Ltd, Tokyo, Japan), and frozen in liquid nitrogen. The tissues were cut into 6 μm thick sections, fixed in acetone, washed in phosphate-buffered saline, and blocked with hydrogen peroxide. The sections were then incubated with anti-MV-nucleocapsid (N) protein pAb, which was produced in our laboratory33 (link), and secondary antibody (Envision HRP kit; Dako). Positive reactions were identified by incubating them with DAB reaction solution. As a negative control, species-matched and filtered sera were used instead of primary antibodies. Images were captured with a Nikon microscope (Nikon, Melville, NY). The sections were fixed for fluorescence microscopy in acetone and stained with Hoechst 33342 (Cambrex Bio Science Walkersville Inc., Walkersville, MD) diluted 1:10,000, and were mounted in Dako Glycerol Mounting Medium. Images were taken on an FV1000 microscope.