Methanol free paraformaldehyde

For immunohistochemistry, cleaned reproductive tracts (see above) were fixed, in toto, in 4% methanol-free paraformaldehyde (Polysciences, Warrington, PA) in mHTF-Hepes overnight at 37 °C. After fixation, reproductive tracts were washed 3x in sterile PBS, air dried at -20 °C for 2–4 hrs, and embedded in Optimal Cutting Temperature compound (Tissue-Tek OCT; VWR, Bridgeport, NJ). Embedded reproductive tracts were stored at -80 °C. For processing, 7-μm sections were cut on a cryostat at −20 °C (CM1850 UV cryostat; Leica, Buffalo Grove, IL), collected on clean glass slides (Diamond White Glass 25 × 75 × 1 mm, (+)- charged; MidSci, Valley Park, MO), and stored at −80 °C until staining. To detect direct GFP expression, slides were brought to room temperature for 5 minutes and stained for 10 min with 10-μg/ml Hoechst 33342. Sections were sealed in antifade solution using a clean coverslip before imaging. In some instances, GFP CETN2 sections were first immunostained using antibodies to Ak15 γ-tubulin (1:500; Sigma-Aldrich) and microtubules (YOL1/34; 1:200; EMD Millipore) as describe above.

Approximately, 2000 HEK293T cells or 3500 U87 cells were seeded in each well of a PDL-coated black optical 96-well μCLEAR-plate (Greiner Bio-One). Transient transfection of HEK293T cells was performed as described above. Twenty four hours after transfection, cells were fixated with 4% [v/v] methanol-free paraformaldehyde (Polyscience Inc) in PBS for 30 min at RT and afterward rinsed with PBS. After blocking with 5% [v/v] FCS in PBS for 30 min at RT, primary antibodies diluted in blocking solution were applied to the cells and incubated over night at 4 °C. Hereby, monoclonal antibodies for hFPR1 (R&D Systems, MAB3744, 1 μg/ml), hFPR2 (Santa Cruz Biotechnology, sc-57141, 0.2 μg/ml), and hFPR3 (R&D Systems, MAB3896, 1 μg/ml) were used. Cells were rinsed three times with PBS and subsequently treated with 2 μg/ml polyclonal alpaca anti-mouse antibody conjugated with Alexa Fluor 568 (Invitrogen) and 2 μM Hoechst 33342 (Thermo Fisher) for 60 min at RT. Image acquisition was performed with a Molecular Devices ImageXpress Micro confocal microscope and analyzed using MetaXpress software (Molecular Devices).