Evos m5000 fluorescent microscope

Seminiferous tubules from the testes of adult mice (90 days old) were used for the in vitro culture experiments. Testes were decapsulated and dispersed with sterile forceps in a Petri dish containing ice-cold medium 199 (Cat. No. 11150059; Thermo Fisher Scientific) with 0.1% BSA for 10 min. The dispersed seminiferous tubules were segmented and placed into the 24-well cell culture plate containing medium 199 and incubated in a CO₂ incubator. After 4 h, 100 μM cyclic peptides were added and incubated for 16 h. Later the media was removed and tubules were washed with 1× PBS. The tubules were kept on a microscopic slide and mounted with SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific) and imaged using EVOS M5000 fluorescent microscope (Thermo Fisher Scientific). Protein samples prepared from treated and untreated seminiferous tubules were run on a PAGE gel and the expression of pGRTH was detected using the phospho-specific GRTH antibody mentioned above. From at least three independent experiments performed, a graph was plotted by calculating the intensity of pGRTH protein band and normalized using β-actin band.