Alexa fluor 488 conjugated wheat germ agglutinin wga

Polydimethylsiloxane (Sylgard 184, Dow Corning) was cured at a 10:1 elastomer to curing agent ratio, manually cut into 1 mm × 1mm × 1 mm blocks, adhered onto 6 mm × 12.5 mm PET frames using Loctite Super Glue Gel Control, and coated with fibronectin sheet as previously described (Supplemental Fig. 2G)[23 (link),48 (link)]. To visualize the samples, fibronectin-coated frames were stained with Alexa Fluor 488-conjugated wheat germ agglutinin (WGA; ThermoFisher), diluted 1:100 in 1× PBS + 0.2% bovine serum albumin + 0.02% sodium azide for 24 hours at 4°C, then rinsed with 0.1% Triton X-100 in 1× PBS (PBST) at room temperature for 30 minutes. Fibronectin-coated frames (n = 2) were loaded onto spring 3 for uniaxial loading and surrounded by PBS during testing. Cyclic loading was performed to 400 μm at a strain rate of 0.04 s⁻¹ for 10 cycles. Tensile test to failure was conducted at 0.04 s⁻¹.

Following treatment with media alone or 5% obese follicular fluid, cumulus cells were plated on coverslips and incubated overnight at 37°C. The cells were fixed with 4% paraformaldehyde for 15 minutes and incubated with 1 μg/mL Alexa Fluor-488 conjugated wheat germ agglutinin (WGA, Thermo Fisher Scientific) to stain the plasma membrane for 30 minutes at 37°C. Cell permeabilization was performed with 0.1% Triton X-100 for 15 minutes. After washing the cells with Dulbecco PBS (DPBS, Corning, Corning, NY), immunostaining for GREM1 was performed with 25 μg/mL goat anti-human/mouse gremlin antibody (R&D Systems) overnight at 4°C. The cells were washed with DPBS and incubated with secondary antibody using 4 μg/mL Alexa Fluor-647 conjugated rabbit anti-goat antibody (Invitrogen) at room temperature for one hour. Following another wash with DPBS, the cells were mounted on ProLong Gold Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific). Imaging was performed with an upright resonant laser scanning confocal microscope (Nikon Fast A1R+, 60x objective). For each woman, we obtained 9–12 images per treatment group. Fluorescence intensity of GREM1 per cell was measured by Icy image processing software.