Confocal microscopy system

Cells were fixed using 4% paraformaldehyde in PBS for 20 min at room temperature before being permeabilized with 0.2% (v/v) Triton X-100 in PBS for 5 min and blocked with PBS containing 1% BSA. Cells were then labeled with primary antibodies anti-vinculin (mouse) antibody (Sigma) (1:100) and phalloidin-TRITC (Sigma) (1:500) in blocking buffer at 4 °C at 37 °C, and washed in PBS. Fluorescent dye (DYE-Light)-conjugated secondary antibodies against goat IgG were used at a dilution of 1:500 for 1 h at 37 °C in blocking buffer. After washing in PBS the samples were mounted with HOECHST 33258 (Sigma), 1 mg/mL in PBS 1× for 5 min. Cells were viewed on a Confocal microscopy system (Olympus) equipped with a 20× (UPlan FLN, NA 0.50), 40× (UPlanFLN, NA 1.30, oil) and 60× (UPlanSApo, NA 1.35, oil) with a resolution of 1024 × 1024 pixels.

To detect the autofluorescence of vitamin A, parts of the livers or pancreas were quickly cut and fixed by paraformaldehyde for 2 hours in total darkness, and 20-μm thick sections were made with a freezing microtome. The sections were examined with Olympus confocal microscopy system (excitation filter BP365/12, barrier filter BP495/40) for the detection of the rapidly fading blue autofluorescence that is characteristic of vitamin A.

Immunohistochemistry was performed as previously described [36 (link), 37 (link), 39 (link)]. Placentas from pregnant mice at E18.5 were harvested, formalin fixed, dehydrated, and embedded in paraffin. Tissue sections, 5 μm in thickness, were cut and deposited on poly-lysine-coated slides. To improve antigen recognition, the sections were microwaved in 10 mM citrate buffer (pH 6.0) for 10~12 min. They were blocked with 10% donkey serum and then incubated with primary antibodies at 4 °C overnight. Primary antibodies were applied in this study: CD117 (R&D Systems, Cat. AF1356, 1:50), cytokeratin 7 (CK7; Abcam, Cat. ab181598, 1:1000), Ki67 (Dako, Cat. M7249, 1:100), CD34 (Abcam, Cat. ab8158, 1:50), CD45 (Abcam, Cat. ab10558, 1:50), CD31 (Abcam, Cat. ab28364, 1:50), vimentin (Abcam, Cat. ab92547, 1:200), E-cadherin (Abcam, Cat. ab53033, 1:50), cardiac troponin I (cTnI, Abcam, Cat. ab47003, 1:200), SPC, and Rho (see “Immunocytochemistry”). Finally, species-matched secondary antibodies conjugated with fluorescence were applied at 37 °C for 1 h. Nuclei were stained with DAPI. A 3,3′-diaminobenzidine system was also carried out for CD117 staining. Images were analyzed using a fluorescence or light microscopy or a confocal microscopy system (Olympus).

Cells were counterstained with rhodamine-conjugated phalloidin to visualize cytoskeletal F-actin filaments and captured using a confocal microscopy system (Olympus) equipped with a 40× (UPlanFLN, NA 1.30, oil) and 60× (UPlanSApo, NA 1.35, oil) with a resolution of 1024 × 1024 pixels. Cell morphology was characterized using the particle measurement feature within ImageJ (www.nih.gov) to obtain spread area, circularity, aspect ratio (A.R.) and Feret’s diameter of single cells. Circularity of cells was calculated as = 4π (area/perimeter²). Values of 1.0 designate a perfect circle, and values near zero are an indication of a more elongated morphology of cells.
Time-lapse imaging was conducted on an Olympus IX73 inverted microscope, equipped with a QImaging OptiMOS sCMOS camera (QImaging, Surrey, BC, Canada) and a stage-mounted incubator with CO₂ and temperature control (H201; Okolab, Pozzuoli, Italy). Bright field images were acquired every 2 min using a 10× (Plan N, NA = 0.25, Ph1) or 20× (LUCPlan FLN, NA = 0.45, Ph2) objective over 8 h. Cell migration was assessed using the manual tracking plugin (mtrackj) for Fiji software. In brief, instantaneous speed was determined by finding the is the length travelled by individual cells divided by time between cell positions in each frame.

Hearts were sectioned at a thickness of 3 μm for immunofluorescence staining. The sections were first blocked with 5% BSA in PBS and then stained with anti‐von Willebrand factor (Abcam Cambridge, MA, USA), anti‐human nuclei antigen (Millipore) and CD8 (Bio‐Rad, Hercules, CA, USA) antibodies. DyLight 488‐ or 594‐conjugated secondary antibodies (Immunoreagents, NC, Raleigh, NC, USA) were used in conjunction with these primary antibodies. Images were taken by a Zeiss (Olympus, Tokyo, Japan) confocal microscopy system. Apoptotic cells in heart tissues were detected by In Situ Cell Apoptosis Detection Kit, FITC (Sangon, Shanghai, China).

Immunocytochemistry was performed as previously described [36 (link)–38 (link)]. Cells were fixed with 4% paraformaldehyde at room temperature (RT) for 10 min, and for intracellular markers, permeabilization of cells was carried out with 0.2% Triton X-100 at RT for 10 min. Next, cells were blocked in 10% donkey serum at RT for 30 min and then incubated in the primary antibodies at RT for 1 h or at 4 °C overnight. Primary antibodies were applied in this study: CD117 (R&D Systems, Cat. AF1356, 1:50), caudal type homeobox 2 (CDX2; Abcam, Cat. Ab76541, 1:200), aquaporin 5 (AQP5; EMD Millipore, Cat. AB15858, 1:100), prosurfactant protein C (SPC; Abcam, Cat. ab40879, 1:100), tight junction protein-1 (ZO-1; Thermo Fisher Scientific, Cat. 40-2200, 1:400), cardiac troponin T (cTnT; Abcam, Cat. ab125266, 1: 200), sarcomeric α-actinin (Sar α-actinin; Sigma Cat. A7811, 1:200), rhodopsin (Rho; Abcam, Cat. ab3267, 1: 200), recoverin (Rec; EMD Millipore, Cat. AB5585, 1: 500), arrestin 1 (Arr1; Abcam, Cat. Ab32099, 1:200). Second antibodies were conjugated with fluorescence, then were incubated with samples at 37 °C for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:1000 in PBS) at 37 °C for 10 min. Images were analyzed using fluorescence microscopy or a confocal microscopy system (Olympus).

SCE assay was performed as previously described^{6 (link),51 (link)}. Briefly, 1 × 10⁶ mouse ESCs were cultured without feeder in ESC medium supplemented with 20 μM bromodeoxyuridine (BrdU, Sigma) for 24 h. In the final 2 h, cells were treated with 0.02 μg/mL colcemid. For SCE assay of mouse NSPCs, cells were cultured in NSPC medium adding 20 μM BrdU for 48 h. In the final 4 h, NSPCs were incubated with 0.02 μg/mL colcemid. Cells were harvested and re-suspended in hypotonic buffer (0.4% sodium citrate and 0.4% KCL) for 15 min at 37 °C. The swollen cells were fixed in freshly prepared methanol-acetic acid fixative (3:1) for 30 min and dropped onto ice-cold wet slides. The slides were dried at 65 °C overnight. The dried slides with metaphase spreads were immersed in 10 μg/mL of Hoeschst 33258 for 20 min and washed with 2 × SSC buffer (0.3 M NaCl, 0.03 M sodium citrate; pH 7.0) once. Then the slides covered with lens paper were exposed coverslip-side-up in 55 °C 2 × SSC buffer to 365 nm UV light at a distance of 1 cm for 30 min. The slides were immersed in 1 × SSC and incubated for 1 h at 50 °C. The slides were stained with 10% Giemsa solution (Gibco, 1892798) for 10 min at 37 °C. After washing with water and dried overnight at room temperature, SCEs images were captured using an Olympus confocal microscopy system.