Vectashield mounting medium with dapi for nuclear counterstain

For immunofluorescent staining of SC35/SRSF2 and UCP1 in primary adipocytes, cells were fixed with cooled ethanol (95%) and glacial acetic acid (5%) (10 min, −20°C), permeabilized by Triton X‐100 (0.3%, 10 min), blocked with donkey serum (10% in PBST with Triton X‐100 (0.3%), 2 h), incubated with primary antibodies against SC35 (mouse anti‐SC35, Abcam ab11826, diluted 1:100 in PBST with Triton X‐100 (0.3%) and donkey serum (1%), overnight) or against UCP1 (rabbit anti‐UCP1, Abcam, diluted 1:200 in PBST with Triton X‐100 (0.3%) and donkey serum (1%), overnight) and fluorophore coupled anti‐mouse or anti‐rabbit secondary antibodies (Alexa Fluor 647 donkey anti‐mouse IgG, Life Technologies or Alexa Fluor 546 donkey anti‐rabbit IgG, Life technologies, both diluted 1:500 in PBST with donkey serum (1%). Slides were mounted with Vectashield Mounting Medium with DAPI for nuclear counterstain (Vector Labs), sealed with a coverslip and nail polish, and visualized with 60× oil immersion objective in a confocal laser‐scanning microscope (Olympus FluoView FV10i). Fluorescent signals were quantified with ImageJ software.

Mitochondria were stained with Mito Tracker Deep Red 633 (100 nM in DMEM, 30 min, 37°C, Invitrogen), fixed with formaldehyde (3.7% in DMEM, 10 min, 37°C), mounted with Vectashield Mounting Medium with DAPI for nuclear counterstain (Vector Labs), sealed with a coverslip and nail polish, and visualized with 60× oil immersion objective in a confocal laser‐scanning microscope (Olympus FluoView FV10i). Fluorescent signals were quantified with ImageJ software.