Alexa fluor 633 conjugated anti mouse igg

BSR cells were seeded on 12 mm diameter round coverslips in 24-well plates and infected with AHSV at a MOI of 2 or 3. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilised with 0.2% Triton X-100 (Merck Millipore) in PBS. After one hour at room temperature in blocking solution (5% milk powder in PBS) coverslips were incubated overnight in primary antiserum diluted 1:100 (anti-NS4) or 1:250 (anti-fibrillarin) in 1% blocking solution. For secondary labeling, cells were incubated with Alexa Fluor-488 conjugated anti-rabbit IgG or Alexa Fluor-633 conjugated anti-mouse IgG (Invitrogen) at a 1:250 dilution. Nuclei were stained with for ten minutes using 5 mg/mL DAPI (4’,6-diamidino-2-phenylindole, Life Technologies) diluted 1:1000 in 1% blocking solution. Coverslips were mounted on microscope slides in VectaShield mounting medium (Vector Laboratories) and the edges sealed. Samples were viewed with a Zeiss LSM510 Meta Laser Scanning Confocal Microscope.

1×10⁴ cells were fixed with 4% paraformaldehyde and 4% sucrose in PBS for 10 min at 4°C. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Cells were treated with 1% BSA in PBS for 30 min at room temperature, and incubated with primary antibodies overnight at 4°C, followed by the treatment with secondary antibodies for 45 min at room temperature. The primary antibodies used in this study were a mouse anti-α-tubulin antibody (1∶1000, DM1A, Abcam), a rabbit anti-ezrin antibody (#3145, 1∶100, Cell Signaling Technology), a rabbit anti-neuronal class III β-tubulin antibody (1∶1000, TUJ1, Covance). The secondary antibodies used in this study were a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Jackson ImmunoResearch) and an Alexa Fluor 633-conjugated anti-mouse IgG (Invitrogen). For filament actin staining, rhodamine phalloidin (Invitrogen) was added to secondary antibody. Fluorescence images were acquired with a confocal laser scanning microscope (FV-1000D, FV-10i, Olympus).

Purified anti-CD3; purified anti-CD28; fluorescein isothiocyanate (FITC)-conjugated anti-CD3, -CD19, -B220, -F4/80, -CD64, -NK1.1, -CD62L, -Helios, -Foxp3, -IFNγ; phycoerythrin (PE)- conjugated anti-CD8, -CD11c, -CD44, -Foxp3, -RORγt, -GATA3, -IL-17F; phycoerythrin -cyanin 7 (PE-Cy7)-conjugated anti-CD3, -CD11b, -CD44, -CTLA-4, -T-bet, -IL-17A; allophycocyanin-conjugated anti-CD4, -CD44, -CD62L, -MHCII, -Foxp3; Brilliant Violet^TM 421 (BV421)-conjugated anti-CD4, -CD45.1, -CD80, -CD86, -IL-10; Brilliant Violet^TM 711 (BV711)-conjugated anti-CD3,-CD45.2, -CD103 (Biolegend or e-bioscience or BD), anti-STIM-1, -NFAT1, -ZAP70, −pZAP70, -SLP76, -pSLP76, -PLCγ, -pPLCγ, -c-Jun, -p-c-Jun, -Myc, -ΕRK, -p-ERK, -HS1, -pHS1; peroxidase-conjugated goat anti-mouse IgG, peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling), Alexa Fluor 633–conjugated anti-rabbit IgG; and Alexa Fluor 633–conjugated anti-mouse IgG (Invitrogen), anti-pPLCγ (Abcam), anti-WASP (Santa cruz), Anti-RIAM (Abcam), and Lpd (Invitrogen), were used for flow cytometry, immunostaining and immunoblotting.