Lamin A/B1 and Adipogenic Marker Staining

For nuclear Lamin A (Abcam, ab8980, 1:400), Lamin B1 (Abcam, ab16048, 1:400), and early adipogenic differentiation marker staining, EP and LP cells from gel and TCP were cultured on collagen-I-coated glass coverslips for 24 h. Cells were then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature (RT) and blocked (3% bovine serum albumin in PBS) for 30 min and washed with cytoskeletal buffer, as described previously (Venugopal et al., 2018 (link)). Cells were incubated with respective primary antibodies for 4 h at 4°C, and then incubated with corresponding secondary antibodies for 1 h at RT. Primary and secondary antibodies were used in the following combinations: anti-PPAR-γ (Abcam, ab59256, 1:300) counterstained with AlexaFluor 488 (Invitrogen, A11034, 1:500), anti-Lamin A (Abcam, ab8980, 1:400) counterstained with AlexaFluor 568 (Abcam, ab175473, 1:400), anti-Lamin B1 (Abcam, ab16048, 1:400) counterstained with AlexaFluor 568 (Abcam, ab175470, 1:400), anti-Vimentin (Sigma-Aldrich, V5255, 1:300) counterstained with AlexaFluor 488 (Invitrogen, A11059, 1:500). Cell nuclei were stained with Hoechst 33342 (Invitrogen, H3570) (1:10,000) in PBS for 5 min at RT and mounted. Images were captured for qualitative and quantitative analysis using EVOS fluorescence microscope (Invitrogen).