Dako antibody diluent

For immunofluorescence staining of tissues, paraffin sections were deparaffinized, and antigens were retrieved by boiling in Dako Antigen Retrieval Agent pH 9 (Dako) for 20 min. After PBS wash, blocking was performed for 1 h with 5% BSA in PBS/PBS⁺⁺ or Dako Antibody Diluent at RT. Primary antibodies diluted in either AB buffer or Dako Antibody Diluent were applied O/N at 4 °C in a humidified chamber. AlexaFluor-conjugated secondary antibodies and DAPI (Invitrogen, Darmstadt, Germany) were applied in Dako Antibody Diluent and incubated for 1 h at RT before washing and mounting in Mowiol. For immunohistochemistry of phosphorylated MLC2 Ser19 (Cell Signaling Technologies, #3675), above protocol was followed, with 5% BSA/PBS⁺⁺ used for blocking non-specific binding sites, and Dako Antibody Diluent used for diluting primary and secondary antibodies plus DAPI. A step-by-step protocol of this procedure has been deposited⁷⁵ .

Upon thawing, tissue sections were fixed with formalin for 30 min. The fixed sections were blocked overnight at 4°C using 0.12 mg/mL unconjugated goat anti-mouse Fab fragment (115-003-007, Jackson Immunoresearch, West Grove, PA, USA) in 0.5% fish skin gelatin (G7765, Sigma-Aldrich) pH 7.4, supplemented with 0.1% Triton X-100. The sections were rinsed 3× with Tris-buffered saline having 0.5% Tween 20 (TBST) for 5 min each. MAb-B43.13 was used as primary antibody at a dilution of 1:6,000 in Dako antibody diluent (S0809, Dako, Glostrup, Denmark) and allowed to incubate overnight at 4 C. The sections were rinsed 3× with TBST and incubated with Alexa Fluor® 488 goat anti-mouse antibody (A-11001, Life Technologies) used as secondary antibody at 1:400 dilution in Dako antibody diluent for 2 h at room temperature. After three washes with TBST, the sections were rinsed with water and counterstained using Hoechst H33342 (2 μg/mL) for 5 min. The sections were rinsed and mounted under a coverslip using FluorSave (345789, Calbiochem). The slides were analyzed with a Zeiss Plan Apochromat 10X/0.45 na lens on a confocal laser scanning microscope (Zeiss LSM 710) using Z-stack for image acquisition. The images were registered using Zen 2011 software (Zeiss) and further processed with Adobe Photoshop CS6.

To analyze cell-free ECM production in HCASMC, an established protocol was used²⁶ . Therefore, cells (5 × 10⁴ cells) were seeded on glass coverslips and cultured overnight until treated with extracellular NLRP3-YFP inflammasome particles (3:1 particles/ cell) for 24 h. As positive control, TGFβ1 (10 ng/ml) was stimulated for 48 h. To exclude potential contamination of ECM extracts with cell surface and intracellular proteins, cells were removed by incubating the wells with ammonium hydroxide (20 mM) for 5 min followed by washing with de-ionized water. Afterwards, ECM was fixed with 2% PFA for 10 min at RT and blocked with 1% BSA/PBS. Antibody against fibronectin (1 µg/ml, Santa Cruz) or IgG mouse isotype control was solved in DAKO antibody diluent (DAKO) and incubated overnight at 4 °C in the dark. Anti-mouse Cy3 antibody (1:1000, Dianova) was diluted in DAKO antibody diluent and incubated for 1 h at RT. To check for successful cell removal, wells were incubated with Hoechst 33,342 (0.5% v/v) in PBS and incubated for 5 min at 37 °C. Cells treated without ammonium hydroxide were run in parallel as positive control. ECM was analyzed on a Keyence BZ-X810 all-in-one fluorescence microscope. Four random selected fields were used for ImageJ analysis. Percentage of positive stained area from images with same magnification was calculated.