Immunostaining and Confocal Microscopy

Confocal microscopy was used to evaluate the effects of LH and AICAR on the presence and regulation of autophagosomes in bovine luteal cells. Mixed luteal cells were seeded at a density of 3 × 10⁵ cells/well on cell culture imaging dishes equipped with #1.5 glass bottom slide and maintained at 37°C in an atmosphere of 95% humidified air and 5% CO₂. Adhered luteal cells were equilibrated in fresh culture medium enriched with 1% BSA for 2 h prior to stimulation with LH (10 ng/ml) or AICAR (1 mM) for 3 h. Organelle specific dyes, CYTO-ID® Green Detection Reagent (autophagosomes), Mitotracker Deep Red (mitochondria), Lysotracker (lysosomes), and LipiBlue (lipid droplets) were added to each well according to manufacturer’s protocol and maintained at 37°C in an atmosphere of 95% humidified air and 5% CO₂ for 30 min. Following incubation, cells were fixed for 30 min with 200 µl 4% paraformaldehyde at 4°C and rinsed three times with PBS.

Images were collected using a Zeiss LSM800 confocal microscope with Airyscan equipped with a 20× (0.8 N.A) and ×63 oil immersion objective (1.4 N.A) and acquisition image size of 1,024 × 1,024 pixel (245.73 µm × 245.73 µm for 20×; 78.01 µm × 78.01 µm for 63×). The appropriate filters were used to excite each fluorophore and emission of light was collected between 450 and 1,000 nm. Cells were randomly selected, and z-stacked (0.3 µm) images were generated from bottom to top of each experiment. To determine the effects of LH or AICAR on mean fluorescence intensity of CYTO-ID® Green Detection Reagent, images were converted to maximum intensity projections and processed utilizing ImageJ (National Institutes of Health) analysis software. Mean fluorescence intensity was determined as previously described (Plewes et al., 2020; Talbott et al., 2020).