Lab tektm 2 chamber slide

Cultured cells were plated in Lab-Tek^TM II chamber slide (ThermoFisher) and fixed in 4% (w/v) paraformaldehyde at room temperature (RT) for 10 min, permeabilized with 0.5% (v/v) Triton X-100 in PBS for 5 min, and blocked in 5% goat serum (Sigma), 3% bovine serum albumin (Fisher), and 0.1% Triton X-100 at RT for 1 h. Primary antibodies were incubated at 4 °C at the indicated dilutions overnight: mouse anti-Flag (1:1000, Sigma) and anti-cGAS (1:1000, Abcam). After three washes in PBS, slides were incubated with indicated secondary antibodies, including goat anti-mouse Alexa Fluor-647 (1:1000, Invitrogen) and goat anti-rabbit Alexa Fluor-488 (1:1000, Invitrogen) at RT for 60 min. After three washes in PBS, slides were mounted in ProLong Gold Antifade Mountant (ThermoFisher). Images were acquired using a confocal microscope (Leica TCS SP8 or Zeiss LSM880).

For enteroid immunofluorescence staining, Matrigel was mechanically disrupted by pipetting, and enteroids were transferred onto Lab-TeK^TM II Chamber Slide (Thermo Fisher Scientific). Enteroids were fixed with 4% paraformaldehyde, followed by permeabilization and blocking with 0.1% Triton X-100 and 5% goat serum in PBS for 30 min at room temperature. Primary antibody reaction was performed with 10 µg/mL Rat monoclonal anti-cryprdin-1 (clone: 77-R63, produced by our laboratory) and 5 µg/mL mouse monoclonal anti-E-cadherin (clone: 36/E-Cadherin, BD Transduction Laboratories) diluted by 1% Triton X-100 and 10% Block Ace (DS Pharma Biomedical) in PBS (antibody dilution buffer) for 2 h at room temperature. Enteroids incubated with Alexa Fluor 488 goat anti-Rat IgG and Alexa Fluor 594 goat anti-Mouse IgG (dilution 1:400, Thermo Fisher Scientific) diluted by antibody dilution buffer for 1 h at room temperature. Enteroids were also counterstained with 5 µg/mL staining 4′6-Diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) for 5 min at room temperature to visualize nuclei and were mounted with Aqua Poly/Mount (Polysciences). Pictures were taken using a confocal microscope (LSM 510 META, Carl Zeiss).