Goat anti gata4

Primary antibodies were those specific to rabbit anti-SALL4 (Abcam, 1:500), goat anti-SOX17 (R&D, 1:500), goat anti-GATA4 (Santa Cruz, 1:300), goat anti-GATA6 (R&D, 1:200), rabbit anti-Nanog (Sigma Aldrich, 1:200). The secondary antibodies used were FITC-conjugated secondary antibodies and TRITC-conjugated secondary antibodies (Jackson ImmunoResearch, 1:200).
Cells were fixed in 4% paraformaldehyde for 15 min at room temperature. Then, removing 4% paraformaldehyde and washing cells with PBS for two times. cells were permeabilized and blocked in PBS containing 0.2% Triton X-100 and 3% donkey serum for 1 h at room temperature. Then the cells were incubated with primary antibodies at 4 °C overnight. After washing three times with PBS, secondary antibodies (Jackson ImmunoResearch) were incubated at 37 °C for 1 h. The nuclei were stained with DAPI (Roche Life Science) for 5 min.

All animal experiments were performed in accordance with Institutional Animal Care and Use Committee of IFOM (project #110/11) approved by the Italian Ministry of Health. Animals were kept on a 12/12-h light/dark cycle (lights on at 07:00 h) with free access to food and water. All animal handlings (sacrifice, etc.) were in accordance with the guidelines established by EU (directive 2010/63/EU). Mice were sacrificed by carbon dioxide euthanasia.
E3.5 embryos were flushed according to standard procedures [36 (link)] and cultured for 24h in M16 (Sigma) medium at 37°C and 0.5% CO2. E4.5 embryos were then fixed in PFA 4%, permeabilized in PBS/0.25% Triton X-100, blocked in PBS/0.05% Triton X-100/3% BSA and incubated with primary antibodies: mouse anti-Cdx2 (Biocare Medical CM226; 1:100), rabbit anti-Nanog (Cosmo Bio RCAB0002P-F, 1:50), goat anti-Gata4 (Santa Cruz sc-1237; 1:100), rabbit anti-cleaved Caspase-3 (Cell Signaling 9661; 1:200), followed by secondary antibodies: donkey anti-mouse A488-conjugated (Invitrogen; 1/100), donkey anti-rabbit CY3-conjugated (Jackson Labs; 1/400), donkey anti-goat CY5-conjugated (Jackson Labs; 1/400), and DAPI (0.5μg/mL) counterstaining. The images were acquired with a TCS SP2 AOBS confocal microscope (Leica Microsystem) and processed with ImageJ software.

A fraction of cultured cells were fixed on days 10, 15, and 20 with 4% polyoxymethylene for 15 min at room temperature. The fixed cells were then permeabilized by using 0.25% triton-X for 20 min, blocked with 5% normal donkey serum (Jackson, USA) for 1 hour, and stained with mouse anti-Ki67 (BD, USA) or rabbit anti-Phospho-Histone H3 (Cell signal, USA) or goat anti-Gata4 (Santa Cruz, USA) at 4 °C overnight. Subsequently, cells were incubated with secondary antibody donkey anti-goat Alexa Fluor 488 (1:500; Invitrogen, USA), or anti-rabbit Alexa Fluor 488 (1:500; Invitrogen, USA) or anti-mouse Alexa 488 (1:500; Invitrogen, USA), at 30 °C for 30 min. Cell nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI). For visual inspection, Nikon Eclipse Ti-S Inverted Fluorescent Microscope was used. For the staining of hHVS (before and after decellularization) and myocardial tissues, they were first fixed with 4% polyoxymethylene, embedded in Tissue-Tek OCT (Sakura Finetek, Japan) and cut into 5 μm sections, and then stained also with chicken anti-GFP or rat anti-CD45 (1:200; Abcam, USA), followed by incubation with secondary antibody Donkey Anti-Chicken FITC (1:500; Abcam, USA) or anti-rat Alexa 488 (1:500; Invitrogen, USA) or/and hematoxylin and eosin (H&E).

Immunostaining assay was performed according to standard protocols. Briefly, the cells were fixed in 4% paraformaldehyde and washed with phosphate-buffered saline (PBS) (Invitrogen, 10010023) and then incubated in PBS containing 0.2% Triton X-100 (Sigma-Aldrich, 9002931) and 0.3% bovine serum albumin (Invitrogen, 11020021). Afterwards, the cells were incubated with primary antibodies including: goat anti-Oct4 (Abcam, ab27985), mouse anti-Nanog (Santa Cruz, sc-374001), mouse anti-Sox2 (Abcam, ab75485), goat anti-GATA4 (Santa Cruz, sc-1237), mouse anti-tyrosine hydroxylase (TH) (Abcam, ab6211), and goat anti- alpha fetoprotein (AFP) (Santa Cruz, sc-8108). The cells were then washed and incubated with goat anti-mouse IgG or rabbit-anti-goat IgG (Invitrogen). A concentration of 0.5 µg/ml diamino phenyl indole (DAPI) (Sigma, 28718903) was used for nuclei staining. Images were visualized using Nikon Ti inverted fluorescence microscope. Alkaline phosphatase (AP) staining was performed using the AP detection kit (Millipore, SCR004).