M1753

cjiPSCs were differentiated in vitro using the EB formation assay according to a previously published protocol (Stauske et al, 2020 (link)). After treatment with collagenase type IV, cell clumps derived from single colonies were cultured as a suspension in UPPS medium for 24 h, to allow complete aggregation. The UPPS medium was then replaced with differentiation medium (IMDM [12440053; Thermo Fisher Scientific], 20% FBS [16000044; Gibco], 1x MEM NEAA [11140-035; Gibco], and 450 μM 1-thioglycerol [M1753; Sigma-Aldrich]). After 8 d, the EBs were transferred to a cell culture plate with coverslips coated with 0.1% gelatine (ø 25 mm; Thermo Fisher Scientific). After 25 d from the start of the protocol, coverslips containing embryoid body outgrowths were fixed and immunostained.

SeeDB protocol was conducted as described by Ke and collaborators8 (link), with modifications. Testes were immersed successively in 20% (w/v) D-(-)-fructose solution (F0127, Sigma-Aldrich) during 1 h, 40% (w/v) fructose during 1 h, 70% (w/v) fructose during 2 h 30 minutes, 100% (w/v) fructose during 4 h and 80,2% (w/w) fructose during 16 h. All steps were performed at room temperature on a rotating wheel. All fructose solutions were prepared in distilled water and contained 0,5% α-thioglycerol (M1753, Sigma-Aldrich).

20%, 40%, 60%, 80%, 100% (w/v) and 80.2% (w/w) fructose (F0127, Sigma-Aldrich) were dissolved in DDW and α-thioglycerol (M1753, Sigma-Aldrich) was added to give a final concentration of 0.5% (SeeDB [standard] solutions). Brain slices were serially incubated in the 20%, 40% and 60% (w/v) fructose solution, each for 4 hr, and then incubated in the 80% and 100% (w/v) fructose solution, each for 12 hr. Afterward, the slices were cleared in the 80.2% [w/w] fructose solution (SeeDB) for 24 hr. All incubations were performed at 20–25°C (Ke et al., 2013 (link)).