Vectashield hardset antifade mounting medium with 4 6 diamidino 2 phenylindole dapi

SRH cells were maintained in osteogenic and adipogenic differentiation media for 21 days. The osteogenic induction medium consisted of DMEM high glucose (4.5 g/L), supplemented with 10% fetal bovine serum, 0.1 μM dexamethasone, 10 mM β-glycerol phosphate, and 50 μM L-ascorbic acid (Merck, Darmstadt, Germany). Adipogenic differentiation medium was purchased from PromoCell (Heidelberg, Germany). Alizarin Red S (Carl Roth, Karlsruhe, Germany) and Oil Red O (Thermo Fisher Scientific, Waltham, MA, USA) were utilized according to the manufacturer’s instructions to depict osteogenic and adipogenic differentiation, respectively.
Intracellular lipid accumulation after adipogenic differentiation was detected by staining with the HCS LipidTOX™ Green neutral lipid stain (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The cells were mounted in VectaShield^® HardSet™ antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA), before being visualized while using an Axio Imager Z1 phase contrast fluorescence microscope (Zeiss, Oberkochen, Germany) that was equipped with an AxioCam MRm camera (Zeiss, Oberkochen, Germany) and AxioVision software v4.8 (Zeiss, Oberkochen, Germany). Cell differentiation and staining was performed in three independent experiments with a representative image shown.

Mice were anesthetized and perfused with heparin sodium salt in phosphate-buffered saline (PBS) and then 4% formaldehyde solution. After post-fixation, the brains were sectioned (60 μm thickness) in a vibratome (Leica VT1200S, Leica, Wetzlar, Germany). Brain sections were mounted with Vectashield Hardset antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, United States). Brain sections were imaged under an A1 HD25 high-resolution confocal microscope (Nikon, Tokyo, Japan) and analyzed using NIS-Elements AR analysis software (Nikon, Tokyo, Japan).