Ihc tek diluent

Synchronized RASMC (seeded at 5 × 103 cells in 96 well glass bottom imaging plate, 655891, Greiner Bio-One) were treated with CA, or RAW 264.7 macrophages pretreated with CA-ARAPas. Treatments were at a ratio of 1:30 RASMC to macrophages. After 24 h, cells were fixed in 2% paraformaldehyde, and permeabilized with 0.1% Triton-X (X100; Sigma-Aldrich, St. Louis, MO, USA). Cells were then probed for Nrf2 (1 µg/mL, ab137550; Abcam, Cambridge, UK) and alpha smooth muscle actin (1:200, 48938; Cell Signaling Technology, Danvers, MA, USA). This followed by AlexaFluor555 goat anti-rabbit IgG (4 µg/mL, A21429; Thermo-Fisher Scientific, Waltham, MA, USA) and AlexaFluor647 goat anti-mouse IgG (2 µg/mL, A21236; Thermo-Fisher Scientific, Waltham, MA, USA). All antibodies were diluted in IHC-Tek diluent (1W-1000; IHC World, Woodstock, MD, USA); nuclei were counterstained with 0.0012 µM DAPI (D3571; Invitrogen). Stained samples were maintained and imaged in PBS. Three-dimensional image acquisition was done using an LSM 780 laser scanning confocal microscope (Carl Zeiss Microscopy, Jena, Germany). Three-dimensional reconstructions were carried out using Imaris v9.5.0 (Bitplane AG, Zürich, Switzerland).