Mount liquid

Gonads were stained as previously reported (Phillips et al, 2009 (link); Gordon et al, 2021 (link)) with the following modifications: for fixative, we used a final concentration of 2% formaldehyde diluted from 16% ampules opened immediately before dissection; in addition, DAPI staining was omitted and MOUNT LIQUID (Abberior) was used as mounting media. STAR RED and Alexa Fluor 594 were used for the two STED channels (see the Antibodies section, below). Images were acquired with an Abberior STEDYCON mounted on a Nikon Eclipse Ti microscope with a 100× 1.45 NA oil immersion objective. Images shown are a single z-section.

To analyze the reorganization of Fn within the films, substrates were built using fluorescently labeled Fn (Fn*) with Alexa Fluor 568. A total of 5,000 HGF/cm² were seeded on the samples using complete medium and incubated at 37°C and 5% CO₂ during 2, 4, and 7 days, respectively, and immunostained as previously described (Section 2.7.5). For STED microscopy experiments, mouse monoclonal anti-vinculin V9131 (Sigma-Aldrich) diluted 1:400 in PBT (PBS + 0.1% Tween 20 + 2% BSA) was used to control focal adhesion formation, and the samples were incubated for 45 min at room temperature. Mouse Abberior STAR RED (Abberior, Göttingen, Germany) diluted 1:200 in PBT was added to the samples as secondary antibody and incubated for 1 h at room temperature. Substrates were washed with PBS (3×) for 15 min and mounted on microscope slides using Abberior Mount Liquid. Samples were examined using Abberior STEDYCON attached to a Zeiss Axio Imager.