Alexa fluor 488 conjugated donkey anti goat igg antibody

For immunofluorescent labeling of TAP-tagged constructs, SK-N-AS cells were seeded out on glass coverslips in a 6-well plate at a density of 40 000 cells/cm² prior transfection. 24 h after seeding, the cells were transfected with 1 μg plasmid DNA using X-tremegene HP (Roche) for 48 h in a ratio of 1:3 (μg DNA:μl X-tremegene HP). After 48 h incubation, transfected cells were washed with 2x2mL of ice cold 1xPBS. Subsequently the cells were fixed and permeabilized with -20°C methanol for 5 min followed by washing with 3x2mL of ice cold 1xPBS. Cells were then blocked in 1x Tris-buffered saline (TBS, 137mM NaCl, 2,7mM KCl, 25mM Tris-Base, pH 7,4) containing 5% bovine serum albumin (BSA) and 1% Tween-20 at room temperature for 1 h. Thereafter, cells were incubated with an Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (Invitrogen, A11055) and DRAQ5 (for DNA staining) at concentrations 1:2000 and 1:5000 respectively, for 1 h at room temperature. Note, TAP-tagged Fe65 was directly detected by the anti-goat antibody due to the protein A domain. The cells were washed 3x500μL/coverslip in 1xTBS containing 1% Tween-20 before mounting on glass slides with Fluoromount-G (SouthernBiotech, 0100–01). Subcellular distribution of immunostained TAP-tagged Fe65 was determined using an imaging system with a Nipcov spinning disc (CSU-22) on a Zeiss Axiovert 200.

Confocal analysis of FXa in platelets was performed as previously described^{16 (link)}. Briefly, resuspended platelets at the concentration of 20 × 10⁶ platelets/ml were cytospinned onto slides, fixed, permeabilized, and stained with a goat anti-human FX antibody (Affinity Biologicals) as the primary antibody, an AlexaFluor 488-conjugated donkey anti-goat IgG antibody (Invitrogen, USA) as the secondary antibody.

Cells were incubated with goat F(ab’)₂ anti-human IgM-coated or control antibody-coated latex beads at a 2:1 bead to cell ratio for 3 h at 37 or 4°C. Cells were then placed on ice for 10 min to terminate internalization. For confocal analysis, 2 × 10⁶ cells were incubated in 1× CellMask™ Deep Red solution (ThermoFisher) as per manufacturer’s instructions for 10 min at 37°C, placed on ice and fixed for 15 min using 4% paraformaldehyde. Samples were then washed and incubated with AlexaFluor488-conjugated donkey anti-goat IgG antibody (20 μg/mL; ThermoFisher A-11055) for 30 min on ice. Cells were washed and cytospun (Thermo Shandon Cytocentrifuge, C4) onto glass slides, dried for 10 min at room temperature and mounted using Vectashield Hardset mountant with 4’,6-diamidino-2-phenylindole (DAPI: Vector Laboratories, H-1500). Images were collected on a Leica SP5 CLSM with a 100× (NA1.4) Plan-apochromatic objective using LAS-AF software (Version2, Leica) and processed using Leica Application Suite X (Leica Microsystems). For flow cytometry, 1 × 10⁶ cells were stained with the AlexaFluor488-conjugated donkey anti-goat IgG antibody (20 μg/mL) for 30 min, washed and then analyzed.