Hoechst h1399

Post-confluent pre-adipocyte cells from day 7 of culture were incubated with vehicle CT (DM ± 0.1% DMSO) or vehicle plus CA (50 μM), HT (100 μM) or RGZ (1 μM), or a combination of CA or HT with RGZ, for 24 h. RGZ was used as a potential rainbow trout peroxisome proliferator-activated receptor gamma (PPARγ) agonist. PPARγ was detected by immunofluorescence, using the protocol described by [32 (link)]. The polyclonal rabbit anti-PPARγ (H-100) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary Alexa Fluor^® conjugated antibody (A21069, goat anti-rabbit 568) was purchased from Life Technologies (Alcobendas, Spain). Nuclei were counterstained with Hoechst (H1399, Life Technologies, Alcobendas, Spain). Images were obtained at 36x magnification on a Leica TCS-SP5 confocal microscope. Nucleus fluorescence was quantified and normalized to the total number of nuclei in the same field, using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cells for immunofluorescence staining were fixed with 4% paraformaldehyde in PBS, and blocked with 5% bovine serum albumin (BSA) in PBS. cTnI (1:250, Abcam) and anti-Goat Cy3 (1:500, Jackson Laboratory) were used as primary and secondary antibodies, respectively. To detect the nuclei, after washing, samples were stained with HOECHST H1399 (1:2,000, Life Technologies). For quantification, the size of cTnI-positive (cTnI⁺) cells from embryo explants were measured by ImageJ (NIH). For YS analysis, dissected embryos were fixed in 2% paraformaldehyde for 20 min, subsequently incubated in 5 and 15% sucrose solutions until embryos decanted and then frozen in 7.5% gelatin. Cryosections were incubated with anti-Jdp2 (1:100, Santa Cruz) and anti-Gata1 (1:100, Santa Cruz) antibodies overnight at 4 °C. Reactions were detected after 1 h of incubation with anti-Rabbit Alexa Fluor 555 (1:400, Invitrogen) and anti-Rat 488 (1:400, Jackson Laboratory). Staining was visualized by laser confocal scanning (Zeiss LM510).

All cells were fixed for 15 min with 4% paraformaldehyde in PBS (Sigma-Aldrich) and permeabilized with 0.5% Triton-X (Sigma-Aldrich) for >4 h at RT before staining. Hoechst (H1399; Thermo Fisher Scientific, Waltham, MA) was used at 5 μg/mL with 15 min incubation at RT to stain nuclei. Primary antibodies were applied overnight at 4°C in a blocking buffer of 5% donkey serum (Sigma-Aldrich) at the following concentrations: anti-laminin (L9393; Sigma-Alrich) 1:500; TRA-1-60 (MAB4360; EMD Millipore, Burlington, MA) 1:100; NANOG (AF1997; R&D Systems) 1:200; CD71 (334107; Biolegend) 1:100.
Secondary antibodies were obtained from Thermo Fisher Scientific and applied in a blocking buffer of 5% donkey serum for 1 h at RT at concentrations of 1:400 to 1:800. A Nikon Eclipse Ti epifluorescence microscope was used to acquire single 10 × images of each micropattern, and a Nikon AR1 confocal microscope was used to acquire 60 × stitched images of each micropattern using the z-plane closest to the micropatterned substrate for reprogramming studies. In brief, EPCs are identified as CD71⁺; Nanog⁻, IMs are indicated as CD71⁻; Nanog⁻, and iPSCs are indicated as CD71⁻; Nanog⁺.