Prolong gold antifade mountant with dapi for nuclear labeling

Experiments were carried out in the ImaFlow core facility (INSERM LNC-UMR1231, Dijon, France). Five-µm sections were deparaffinized and antigen retrieval was performed in 95 °C citrate buffer. Sections are left to return at room temperature and unspecific binding was blocked with PBS—2% BSA—0.5% Tween 20. Primary antibody anti-IL-1β was incubated (1/50 dilution in the blocking solution) overnight at 4 °C followed by secondary antibody staining Alexa Fluor™ Plus 555-conjugated goat anti-mouse (1/1000). Slides were mounted in a ProLong™ Gold Antifade Mountant with DAPI for nuclear labeling (P36931, ThermoFisher Scientific, Waltham, MA, USA) and were observed with an Axiozoom epifluorescence microscope (ZEISS, Rueil Malmaison, France). Five representative pictures from three independent experiments were taken in random chosen fields and fluorescence was quantified by measuring mean fluorescence intensity using Zen Software (ZEISS, Rueil Malmaison, France). Results were expressed as means ± standard error of the mean (SEM) in arbitrary density units (ADU).

Myometrial cells (2.5 × 10⁴) were plated on coverslips in 24-well dishes and treated as previously described. Cells were fixed in 4% formaldehyde for 5 min followed by an incubation in permeabilization-blocking buffer for 30 min (0.1% saponin, 3% BSA, 0.1% Tween 20 in PBS). Actin and vinculin were visualized using Alexa Fluor 488 phalloidin (1/400) and anti-vinculin (1/100) with Alexa-fluor 555-conjugated goat anti-mouse IgG antibody (1/1000) as a secondary antibody. Coverslips were rinsed twice in PBS and mounted in a ProLong™ Gold Antifade Mountant with DAPI for nuclear labeling (P36931, ThermoFisher Scientific, Waltham, MA, USA). The slides were observed with an Axiozoom epifluorescence microscope (ZEISS, Rueil Malmaison, France). Five representative pictures from four independent experiments were taken in random chosen fields and fluorescence was quantified by measuring mean fluorescence intensity using Zen Software (ZEISS, Rueil Malmaison, France). Results were expressed as means ± SEM in ADU.