Donkey anti goat alexa fluor 488 antibody

A total of 1 μg of recombinant Salmonella PagC, Rck, and FimA proteins (75 (link)) were subjected to 15% SDS-PAGE. Proteins were electrotransferred onto a nitrocellulose membrane and blocked with 3% bovine serum albumin (BSA). Subsequently, the membrane was incubated with purified human FH (1 μg/ml, in 0.1% BSA), which is below the physiological concentration of FH in serum (500 μg/ml) (76 (link)). Binding of the protein was detected by immunoblotting with goat anti-human FH and donkey anti-goat HRP-conjugated secondary antibody as described above. For immunolabelling the OMVs, purified OMVs from the quadruple mutant induced to express Rck or PagC from corresponding plasmids were incubated with 2 μg of purified human FH, in a final volume of 200 μl. The OMVs were then washed with sterile PBS at 200,000 × g (Type 42.2 Ti rotor) for 15 min at 4°C followed by incubation with anti-human Factor H antibody (1:1,000 dilution) and a 1:2,000 diluted donkey anti-goat Alexa Fluor-488 antibody (Abcam, Cambridge, UK) as the labeled secondary antibody. The fluorescently labeled OMVs were then analyzed by fluorescence NTA (F-NTA) using Zetaview.

Ten-micrometer sections of frozen heart tissue were incubated at room temperature with primary anti–IL-36R, anti–IL-36α, anti–IL-36β, or IgG control antibodies (1:100 dilution; R&D Systems, polyclonal) and a secondary donkey anti–goat Alexa Fluor 488 antibody (1:100 dilution; Abcam, polyclonal). Sections were also incubated with a PE anti–mouse CD31 antibody (1:100 dilution, BioLegend, clone 390), an Alexa Fluor 647 anti–mouse CD106/VCAM-1 antibody (1:100 dilution, BioLegend, clone 429), or an anti-DNA/RNA damage antibody to detect oxidative damage (1:100 dilution, Abcam, clone 153A). Images were captured using an EVOS FL (Thermo Fisher Scientific) or multiphoton microscope (FVMPE-RS, Olympus). ImageJ was used to quantify the MFI of each image with additional analysis of MFI on regions containing only coronary capillaries or a large blood vessel.

To detect the expression of ACE2 on cell surface, Vero-E6 cells, Calu-3 cells, A549 cells, HEK293T cells, and Hela cells were seeded onto 6-well plates and harvest by treatment with 0.25% trypsin (without EDTA). The cells were fixed with 3% paraformaldehyde at room temperature for 15 min, then washed three times with FACS wash buffer (PBS containing 2% FCS), and incubated for 2 h with ACE2 antibody (R&D system, AF933) or IgG isotype antibody (Abcam), which served as a control. Cells were then washed and stained with donkey anti-goat Alexa Fluor 488 antibody (Abcam) for 1 h. All cells were analyzed by using an Apogee flow cytometer.
To detect the expression of ACE2 in cells after inhibitor treatment, Vero-E6 cells were treated with diltiazem (150 μM) or BAPTA-AM (25 μM) for 1 h or CACNA1C-silenced for 72 h, then the cells were harvested as described above, and permeabilized with 0.1% saponin (Sigma) for 20 min, before being incubated with an ACE2 antibody to detect the expression of ACE2 as above.