Mouse anti ha tag

Primary mouse cerebellar Purkinje cells were fixed in 4% paraformaldehyde for 30 min at room temperature. All reagents were diluted in 100 mM phosphate buffer (PB), pH 7.3. Fixed cells were incubated with primary antibody diluted in blocking solution (PB + 3% non-immune goat serum + 0.5% TritonX-100) for 1 h at room temperature. After washing with PB, the corresponding fluorescence-conjugated secondary antibodies were added to the cells in PB containing 0.1% Triton X-100 for 2 h at room temperature. The following primary antibodies were used: rabbit anti-Calbindin D-28K (1:500, Swant, Marly, Switzerland); mouse anti-Calbindin D-28K (1:500, Swant, Marly, Switzerland); Guinea pig anti-Calbindin (1:4000, SYSY, Göttingen, Germany); rabbit anti-GFP (1:2000, Novus, Zug, Switzerland); chicken anti-GFP (1:2000, Abcam, Cambridge, United Kingdom); and mouse anti-HA tag (1:1000, Invitrogen, Massachusetts, United States). The staining was visualized with anti-mouse Alexa 568, anti-guinea pig Alexa 568, anti-chicken Alexa Fluor Plus 488, and anti-rabbit Alexa 488 secondary antibodies (1:2000, Molecular Probes, Eugene, OR, USA). The stained cells were mounted with Mowiol (Sigma-Aldrich, Buchs, Switzerland). The images were captured on an Olympus AX-70 fluorescence microscope equipped with a Spot Insight digital camera.

The following antibodies were used in this study: mouse monoclonal anti-TBEV E (United States Army Medical Research, Institute of Infectious Diseases, Fort Detrick, Frederick, MD, USA), mouse monoclonal anti-TBEV prM (United States Army Medical Research), mouse monoclonal anti-TSG101 (Santa Cruz), mouse J2 anti-dsRNA (SCICONS, Hungary), mouse monoclonal anti-ALIX (Santa Cruz), mouse monoclonal anti-CHMP4A (Santa Cruz), rabbit anti-TSG101 (Atlas antibodies), rabbit anti-CHMP4A (Invitrogen), rabbit anti-ALIX (Atlas antibodies), rabbit anti-HA tag (Invitrogen), mouse anti-HA tag (Invitrogen), rapid anti-GRP94 (Invitrogen), mouse anti-GAPDH (Sigma), mouse anti-βactin (Sigma), mouse monoclonal anti-mCherry tag (Novus Biological), rabbit monoclonal anti-mCherry tag (Novus Biological), Alexa Fluor 594-conjugated anti-mouse goat antibody (Invitrogen), Alexa Fluor 488-conjugated anti-rabbit goat antibody (Invitrogen), anti-mouse VisUCyte HRP Polymer (R&D Systems, USA), and HPR-conjugated anti-mouse goat antibody (Invitrogen).

Cells from different groups were lysed with 1 × SDS sample buffer, boiled for 10 min and separated by 12% SDS-PAGE, and were then transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). After blocking for one night at 4°C with 10% skim milk, the membranes were incubated with diluted primary and secondary antibodies for 2 and 1 h at room temperature, respectively. The result was detected with Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA). The antibodies were as follows: Mouse anti HA-tag (1:1000; Invitrogen, Carlsbad, CA, USA), mouse anti-SOCS3 (1:500; polyclonal antibody prepared in our laboratory), mouse anti-HN (1:1000; monoclonal antibody prepared in our laboratory), rabbit anti-phospho-STAT1 (1:500; ZEN BIO, Chengdu, China), mouse anti-STAT1 (1:500; ZEN BIO, Chengdu, China), rabbit anti-ERK1/2 (1:500; ZEN BIO, Chengdu, China), rabbit anti-phospho-ERK1/2 (1:500; ZEN BIO, Chengdu, China), mouse anti-β-Tubulin (1:2000; Sungene Biotech, Tianjin, China) and goat anti-mouse (1:5000; Sungene Biotech, Tianjin, China).