Alexa fluor 488

For immunofluorescence, cells were fixed at room temperature using PTEMF (20 mM PIPES, pH 6.8, 10 mM EGTA, 1 mM MgCl₂, 0.2% Triton X-100 and 4% paraformaldehyde) for 10 min and permeabilized at room temperature in 0.5% Triton-X100 in phosphate-buffered saline (PBS) for 10 min. Cells were blocked in 3% BSA in PBS for 30 min. Cells were incubated for 1 h at room temperature with primary antibodies as follows: rabbit anti-α-tubulin (PA5-19489, Invitrogen; 1:1000), mouse anti-α-tubulin (B-5-1-2; Sigma; 1:1000), mouse anti-CHC (X22; CRL-2228, ATCC; 1:1000), rabbit anti-CKAP5 (PA5-59150, Thermo Fisher Scientific; 1:400), rabbit anti-chTOG (Fig. 7 only; 34032, QED Biosciences; 1:5000), mouse anti-TACC3 (ab56595, Abcam; 1:1000), mouse anti-GTSE1 (H00051512-B01P, Abnova; 1:1000) and rabbit anti-PIK3C2A (22028-1-AP, Proteintech; 1:1000). Cells were washed three times with PBS, then incubated for 1 h with Alexa Fluor 568- or Alexa Fluor 647-conjugated secondary antibodies (Molecular Probes). Finally, coverslips were rinsed with PBS and mounted with Mowiol containing DAPI (Sigma). In some experiments it was necessary to boost the GFP signal of the knock-in cells. To do this, GFP-booster (Alexa Fluor 488, Chromotek) or GFP rabbit anti-Tag, Alexa Fluor 488 (Invitrogen) 1:200 was used during the primary incubations.

Immunohistochemical staining was performed in chorionic villous sections (n = 5) as previously described^{27 (link)}. Briefly, after deparaffination and rehydration, the sections were boiled in 10 mM sodium citrate buffer for antigen retrieval and permeablized in PBS containing 0.4% Triton X-100. After blocking with goat serum and subsequent incubation with primary antibodies (Table 1), the sections were incubated with Alexa Fluor 488- or Alexa Fluor 594-labeled secondary antibodies (Proteintech, Rosemont, IL). Nuclei were stained with DAPI (1 μg/mL). The staining signals were examined under a fluorescence microscope (Zeiss, Germany).Table 1

Information of the antibodies for immunohistochemical staining and western blotting.

Target protein	Company	Catalog No.	Applications
PDK4	Proteintech	12949-1-AP	WB (1:1000), IF (1:200)
β-hCG	Thermo Fisher Scientific	MA5-14375	IF (1:100)
SPINT1	Santa Cruz	sc-137159	IF (1:200)
11β-HSD2	Santa Cruz	sc-20176	WB (1:2000)
α-Tubulin	Proteintech	66031-1-Ig	WB (1:5000)
PDHE1α	Abcam	ab110334	WB (1:1000)
pPDHE1α	Novus	NB110-93479	WB (1:1000)
Rabbit IgG	Proteintech	30000-0-AP	WB and IF

A suspension of P3 generation UC-MSCs was collected, and the density was adjusted to 1×10⁶/ml, after which the following monoclonal fluorescent antibodies were added: CD31-ECD, CD34-ECD, CD45-ECD, HLA-DR-ECD, CD29-ECD, CD90-ECD, CD73-ECD and CD105-ECD (Bio Legend). Then, the cells were incubated in the dark at 4°C for 30 min, washed twice with PBS and resuspended. The expression of related antigens was detected by flow cytometry.
HDFs were identified by IF. Furthermore, IF was also used to investigate the activation of fibroblasts after 24 hours of stimulation by each group of stem cell supernatant. Briefly, the cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton^™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The HDFs were labeled with vimentin antibody (Abcam, Alexa Fluor 594) at a 1:500 dilution in 0.1% BSA, and myofibroblasts were labeled with α-SMA antibodyat (Proteintech, Alexa Fluor 488) a 1:200 dilution in 0.1% BSA, and incubated at 4°C overnight. Nuclei were stained with DAPI.

The hearts were harvested, washed with PBS, and fixed in 4% paraformaldehyde (Biosharp, Cat No. BL539A) at 4°C for 24 h, followed by incubation in 30% sucrose and then embedded in OCT at −80°C. Frozen sections (8-μm thickness) were processed and permeabilized using 0.3% Triton X-100 (Solarbio, Cat No. T8200) for 30 min, and then blocked with 1% bovine serum albumin (BioFroxx, Cat No. 4240GR) in PBS at 37°C for 1 h. The sections were incubated with primary antibodies: anti-TNNT2 (1:100, Abcam, Cat No. ab8295), anti-KMT2D (1:100, Merckmillipore, Cat No. ABE 1867), secondary antibody (1:500, Alexa Fluor 488, Proteintech, Cat No. SA00013-2; Alexa Fluor 568, Invitrogen, Cat No. A11031). The nuclear staining was performed with DAPI (Beyotime, Cat No. C1005). Immunofluorescences images were visualized using an OLYMPUS microscope BX43.

Different treated HCC cells were cultured and fixed on 14 × 14 mm glass slides. After first incubated with antibodies specific for E‐cadherin (Bioword), ZO‐1 (Proteintech), N‐cadherin (Bioword), slug (Bioword), twist (Bioword), vimentin (Bioword) and then with goat anti‐rabbit IgG (Alexa Fluor 488; Proteintech), the slides were mounted by adding DAPI Fluoromount‐G (Beyotime) and examined with an Eclipse Ti‐S inverted phase/fluorescent microscope (Nikon, Tochigi, Japan).

Primary rat VSMCs and arterial sections were collected for immunofluorescence staining. Briefly, VSMCs were fixed in 4% paraformaldehyde for 30 min, and arterial sections were repaired with antigen retrieval sodium buffer. Cells or arterial sections were permeabilized with 0.4% Triton X-100 (catalog no.: ST795; Beyotime) and blocked with 10% donkey serum (catalog no.: SL038; Solarbio, China) at room temperature. Samples were incubated with primary antibodies against RGMa (1:50 dilution; catalog no.: sc-393046; Santa Cruz), CD68 (1:100 dilution; catalog no.: ab201340; abcam, UK), SM22α (1:200 dilution; catalog no.: ab14106; abcam), α-SMA (1:200 dilution; catalog no.: 19245; CST, UK), and Slug (1:50 dilution; catalog no.: sc-166476; Santa Cruz) at 4°C overnight, followed by secondary antibodies Alexa Fluor 488 (1:200 dilution; catalog no.: SA00013-5; Proteintech, China) and 555 (1:100 dilution; catalog no.: A0460; Beyotime) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (catalog no.: C1002; Beyotime) for 10 min and protected by antifade mounting medium. Triple immunofluorescence staining was performed according to the manufacturer's instructions (catalog no.: AFIHC024; China). Images were captured using a confocal laser scanning microscope (Andor, UK) and analyzed by ImageJ software.