Zenon kit

Monocytes were stained with mouse monoclonal antibodies against CD14-PE (BD Pharmingen), BFRF1 [18 (link)], gp-350-220 [13 (link)], rabbit TLR8 (Cell Signaling), and secondary-antibodies-Cy3-conjugated (Jackson IR, West Grove, PA, USA), Alexafluor-350 goat-anti mouse antibody (Invitrogen, Grand Island, NY, USA), or 488-labeling (Zenon kit; Invitrogen). Coverslips were mounted using Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA) and immunofluorescence staining was examined using a FluoView FV10i confocal microscope system (Olympus, Center Valley, PA, USA) at 488 nm (green), 594 nm (red), and 405 nm (blue). Paraffin sections of skin tissues were stained using mouse mAb against CD163 (AbD Serotec, Raleigh, NC, USA) and Zta/Zebra (Argene, bioMérieux, Inc., Durham, NC, USA) as previously described [13 (link)]. Immunohistochemical staining was examined using the Olympus BH2 microscope.

REF52 YFP-paxillin stable cell line (kindly provided by Benjamin Geiger and Joachim Spatz) were cultured in DMEM (PAN Biotech, Aidenbach, Germany) supplemented with 1% glucose, 10% fetal calf serum, 1% nonessential amino acids and 1% L-glutamine and maintained at 37°Cand 5% CO₂. The primary antibodies included anti-vinculin mouse IgG1, anti-zyxin rabbit IgG, anti-α-actinin mouse IgM (V9264, Z4751, A5044; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), anti-paxillin-pY118 rabbit IgG, anti-FAK-pY397 rabbit IgG (44-722G, 44-624G; Invitrogen GmbH, Karlsruhe, Germany), anti-VASP rabbit IgG (3132, Cell Signaling Technology, Frankfurt, Germany), Alexa Fluor 555 conjugated anti-FAK mouse IgG1 (clone 4.47, 16-234, Merck Millipore, Darmstadt, Germany), TRITC conjugated anti-paxillin mouse IgG1 and anti-Hic-5 mouse IgG1 (610055, 611164; BD Transduction Laboratories). F-actin was labeled with Alexa Fluor 350 Phalloidin (A-22281, Invitrogen GmbH). A secondary antibody for α-actinin staining was Alexa Fluor 350 goat anti-mouse IgM (A-31552, Invitrogen GmbH). The other unconjugated primary antibodies were pre-labeled with Zenon kit (Z-25041, Z-25306, Z-25006, Z-25308, Z-25008, Invitrogen GmbH) according to manufacturer protocol.

The frozen tissue sections were fixed in −20°C acetone for 10 min and washed in phosphate-buffered saline (PBS). Background was blocked with 2% bovine serum albumin, 0.3% Triton-X100, and 5% goat serum (Invitrogen, Carlsbad, CA) in PBS for 30 min at room temperature (RT). Three primary antibodies were combined in each protocol, diluted according to Supplementary Table S2. Primary antibodies were incubated at 4°C overnight. The sections were washed, and the following secondary antibodies were added for 1 h at RT: goat anti-mouse Alexa Fluor 546, goat anti-rabbit Alexa Fluor 546, or goat anti-rabbit Alexa Fluor 647 (Invitrogen). To enable the use of two mouse primary antibodies, the cTnT antibody was conjugated with Alexa 488 fluorochrome using Zenon Kit (Invitrogen). The samples were fixed using Histofix (Histolab, Gothenburg) for 15 min, washed, and mounted with prolong gold antifade reagent with nuclei staining 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Corresponding isotype controls for primary antibodies and did not show specific staining.

Cells were cultured on glass cover-slips, fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton-X100 and immunostained with the indicated primary antibodies. Subsequent visualization was performed with AlexaFluor-conjugated Ab (Life technologies). For ICAM-2/VECad or VECad/NCad co-staining, VECad was labeled using the ZENON® kit (Life technologies), according to the manufacturer’s protocol. Nuclei were visualized using TOPRO-3 (Life technologies). Images were captured with a confocal microscope (LSM510 META; Carl Zeiss). Adobe Photoshop was used according to the guidelines to construct the confocal multi-channel images, to select specific regions of interest and to apply minor alterations to contrast and brightness uniformly across the entire figure panel.

For surface staining, cells were infected or treated as indicated and washed with PBS, incubated in blocking buffer (10% rabbit IgG or FBS in PBS) and stained with relevant antibodies. For analysis of intracellular and cell-associated bacteria, GAS cultures were washed, and the bacteria labeled with an Alexa Fluor 660 NHS Ester (Life technologies). The following antibodies and isotype controls were used: anti-mouse CD38-FITC (BD Biosciences, cat #558813, clone 90), rat IgG2a isotype control-FITC (eBioscience, cat #11-4321-82, clone eBR2a), anti-Streptococcus A-FITC (LSBio, cat #LS-C86701), anti-P2X7 receptor (extracellular) (Alomone, cat #APR-008), purified rabbit IgG isotype control (Life technologies, cat #026102). A Zenon kit (Life technologies) was used to label the P2X7 antibody and the purified rabbit IgG with the fluorescent conjugate Alexa Fluor 647. Stained cells were washed and fixated with 4% paraformaldehyde (PFA), acquired on a BD LSR II or Accuri C6 flow cytometer and analyzed using FlowJo.