Alexa fluor 488 conjugated goat anti mouse igg h l secondary antibody

The samples were incubated with 1:100 diluted anti-activated integrin β1 antibody (Sigma-Aldrich, MAB2079Z) in HEPES-Buffered Hanks Balanced Salt Solution (HHBSS), for 30 min at 4 °C. The samples were then washed with ice-cold HHBSS for 3 times and fixed with 4% Paraformaldehyde in PBS for 15 min at room temperature (RT). The fixed samples were washed with PBS for 15 min and incubated in blocking buffer (5% Bovine serum albumin in PBS) for 1 h at RT. The sample were then incubated with 1:500 Alexa Fluor 488-conjugated goat anti-Mouse IgG (H + L) secondary antibody (Thermo Fisher Scientific, A11001) in blocking buffer at RT for 45 min, washed with PBS for 15 min at RT. The experiments were performed on 2 devices and images were taken of 59 pads in total. Among these, 44 out of the 59 imaged pads exhibit similar effect as shown in the representative images in Fig. 1i-k.

For confocal laser scanning microscopy, 10⁵ moDC were incubated at 37°C for 2 h on 6 channel μ-Slide (Ibidi) and subsequently stimulated for 4 h with 10⁷ M. stadtmanae cells. After fixation in 3% paraformaldehyde, primary antibodies were added at 1:100 in PBS with 3% BSA and 0.1% saponin and incubated overnight at 4°C. Cells were incubated with anti-NF-κB p65 (1:100, F-6), anti-IRF1 (1:100, B-1), or anti-IRF5 (1:100, H-56, all from Santa Cruz Biotechnology) overnight at 4°C, followed by staining with an Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:300, Invitrogen). The nuclei were counterstained using Hoechst 33342 dye (1:3,000, Life Technologies). For detection of ASC specks, BLaER1 cells were stimulated for 4 and 18 h, and staining was performed as previously described. ASC (1:100, B-3, Santa Cruz Biotechnology) was used as the primary antibody, and Alexa Fluor 546-conjugated goat anti-mouse IgG (H + L) was used as the secondary antibody (1:300, Invitrogen). Images were captured using the TCS SP5 confocal microscope and LAS AF software (both from Leica).

Titers of viral stocks were determined by indirect immunofluorescence assay (IFA) using MDCK cells. Ten-fold serially diluted virus samples with TPCK-trypsin (1 µg/mL) were added to MDCK cell monolayers grown in a 96-well tissue culture plate. After 24 hr of incubation, IFA was performed as described previously [18] (link). Briefly, cell monolayers were washed once in PBS, fixed with 100 µL/well of 80% acetone in milli Q water for 10 min, liquid discarded, plates dried in the fume hood for approximately 30 min, and finally the cells were soaked in PBS-0.05% Tween 20 (PBS-Tween) for 5 min. Cells were subsequently incubated with IAV nucleoprotein specific monoclonal antibody (Cal Bioreagents, M058) (1∶5,000) at 37°C for 2 hr. After washing with PBS-Tween, Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, A11029) (1∶4,000) was added and incubated for 1.5 hr. Stained cells were washed with PBS-Tween and preserved with a mounting medium (60% glycerol in PBS). Cells were examined for the presence of fluorescent-staining cells using an Olympus IX51 microscope with a FITC wide pass filter set. The viral titer was calculated using the Reed and Muench method and expressed as TCID₅₀ per mL as described previously [18] (link), [19] . The number of fluorescent focal units (FFU) per mL was then calculated as TCID₅₀ per mL [20] (link) (Table 1).