Alexa flour 647 goat anti rabbit igg

The cell morphology was observed at different time points after infection using Ph Microscopy by Nikon inverted microscope Ti-U (ECLIPSE, Japan). hBMECs were seeded at a density of 1.5 × 10⁴ per well on chamber slides and deposited at the bottom of a 24-well plate. The cells were inoculated with A/WSN/33 (H1N1) at 0.1 MOI and incubated at 37°C at 5% CO₂ for 12 h. The slides were rinsed three times with 1 × PBS and further fixed with 4% paraformaldehyde (PFA) for 10 min at 37°C. After washing with PBS, the cells were permeabilized in 0.1% Triton X-100 in 1 × PBS at room temperature for 10 min, then washed again with PBS. Permeabilized cells were then blocked with 2% BSA in PBS for 1 h at room temperature and washed. For immunofluorescence, the cells were incubated with the primary anti-nucleoprotein NP (1:500; rabbit polyclonal, GeneTex Inc., CA, USA) diluted in 0.1% BSA and incubated for three hours at room temperature. The secondary antibody used was Alexa flour 647 goat anti-rabbit IgG (1:500; Thermofisher). After washing, the cells were stained for F-actin with fluorescent FITC-conjugated Phalloidin for 2 hat 37 °C and with Hoechst for 5 min. The slides were observed under a laser confocal microscope (Leica, Germany).

Cells were grown on 8-well Lab-Tek chamber slides (Thermo Scientific) and fixed 24 h after transfection with paraformaldehyde (4%) in phosphate-buffered saline (PBS) for 10 min at room temperature (RT). Fixed cells were washed with PBS twice and permeabilized with 0.2% Triton X-100 for 5 min at RT, blocked with 5% FCS, and incubated with anti-HA rabbit antibody (1/100) or anti-BALF0/1 rabbit sera (1/200) for 1 h at 37 °C. Then, the cells were washed with PBS and incubated with the secondary antibody at a dilution of 1/1000 (Alexa Flour 555 goat anti-rabbit IgG or Alexa Flour 647 goat anti-rabbit IgG, Thermo Scientific). Next, the cells were washed with PBS and the nuclei were counterstained with Hoechst 33342 (Thermo Scientific). Coverslips were mounted in Glycergel mounting medium (Dako) and observed by using a Zeiss AxioObserver Z1 or Leica SP8 confocal laser microscope. Images were resized, organized, and labeled using ImageJ software. Three-dimensional reconstruction was established by IMARIS (Bitplane, Belfast, UK) software.

First, 5 × 10³ cells/chamber HAP1 cells were cultured in an 8-well slide and chamber (192-008, Watson, Tokyo, Japan). Cells were fixed with 4% PFA in PBS (26123-55, Nacalai Tesque), washed with PBS three times, and blocked with blocking buffer (5% skim milk with 0.3% triton X-100). Primary antibodies (anti-TLS/FUS (ab154141, Abcam, Waltham, MA, USA), anti-ZAP3 (A304-038A, Bethyl, Montgomery, TX, USA), anti-Matrin3 (A300-590A, Bethyl)) were diluted with blocking buffer at 1:1000, anti-TIA-1 (ab140595, Abcam) and anti-TDP-43 (12892-1-AP, Proteintech, Rosemont, IL, USA) were diluted at 1:250, and incubated at 4 °C for 16 h. Cells were washed for three times, and incubated with secondary antibodies (AlexaFlour 647 goat anti-rabbit IgG (A21244, Invitrogen) and Alexa Fluor 555 donkey anti-mouse IgG (A31570, Invitrogen), diluted with blocking buffer at 1:1000) at 37 °C for 1 h. Cells were washed again and mounted with Vectashield (H-1800, Vector Laboratories, Burlingame, CA, USA), and the cells were observed under fluorescent microscopy (BZ-X700).