Anti collagen 4

Antibodies used for immunofluorescence staining: anti-CD31 (Dako, Mouse, Cat# M0823, RRID AB_2114471), anti-CD11b (Sigma-Aldrich, Rabbit, Cat# SAB5600105, RRID:AB_2910138), anti-Collagen IV (Bio-Rad, Rabbit, Cat# 2150-0140, RRID:AB_2082644), anti-Hif1α (Bethyl (Thermofisher), Rabbit, Cat# A300-286A, RRID:AB_2117114), Cleaved-Caspase 3 (Cell Signaling, Cat# 9661, RRID:AB_2341188). Secondary antibodies: goat anti-mouse-Cy5 (Jackson ImmunoResearch, Cat# 115-175-166, RRID:AB_2338714), goat anti-rabbit-Alexa Fluor 750 (Thermofisher, Cat# A-21039, RRID:AB_2535710). Dilutions are listed in Supplementary Table 5.
Antibodies used for FACS-analysis: anti-ALDH1A1-PE (SinoBiological, Cat# 11388-MM03-P, RRID:AB_2860344) and anti-CD44-Alexa Fluor 700 (ThermoFisher, Cat# 56-0441-80, RRID:AB_494012).

The shControl or shPdgfd ESCs (1 × 10⁶ cells/100 µL PBS) were injected subcutaneously into the dorsal flank regions of nude mice. Four weeks after injection, the teratomas were surgically dissected and weighed. Parts of the tumors were used for RNA extraction, and the rest were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (5 µm) for immunohistochemistry (IHC) staining.
For IHC staining, sections were placed at 60 °C for 1 h followed by deparaffinized with xylene and ethanol. Subsequently, the sections were processed in 10 mM citrate buffer (pH 6.0) and boiled for 10 min for antigen retrieval. After cooling in room temperature, the sections were incubated in 3% hydrogen peroxide for 10 min to block the endogenous peroxidase activity. The sections were stained with anti-Collagen IV (BioRad, Hercules, California, USA) overnight at 4 °C and then incubated using the Elivision kit (Maxim Biotech, Fuzhou, China) for 30 min at room temperature. The DAB color reagent (Maxim Biotech, Fuzhou, China) was used. Finally, hematoxylin staining was performed for nuclear visualization. Sections were visualized using a fluorescence microscope-Axio Imager Z2 (ZEISS, Oberkochen, Germany). ImageJ software was used to analyze the sections. Collagen IV density was calculated as Collagen IV⁺ pixels/total field pixels.