Ab96881

Macrophages were washed with PBS three times. Four percent fixative solution (Solarbio, P1110) was added to the Petri dish for 10 min. Then, the solution was removed and cells washed three times. Next, 0.5% Triton X-100 (Solarbio, T8200) was added to the dish for 10 min. The solution was removed and cells washed three times. Five percent BSA (Solarbio, A8010) was added to the dish, and it was incubated for 1 h at room temperature. Then, primary antibodies were added to the M0 (GLRX1: 1:500, Abcam, ab45953; CD11b: 1:100, Proteintech, 66519-1-lg) and M2 macrophages (GLRX1: 1:500, Abcam, ab45953; CD163: 1:100, Abcam, ab156769) (18 (link)), respectively, and they were incubated overnight at 4°C. The solution was removed and cells washed three times. Secondary antibodies (DyLight 488 goat antirabbit polyclonal antibody, Abcam, ab96899, 1:200; DyLight 594 goat antimouse polyclonal antibody, Abcam, ab96881, 1:200) were used for 1 h at room temperature. The solution was removed and cells washed three times. Prolong™ Diamond Antifade Mountant with DAPI (Invitrogen, P36962) was added to the dish, and photos were taken with confocal microscopy.

After treatment, the samples (cells cultured on glass coverslips and frozen mouse skin sections) were fixed in 4% paraformaldehyde for 20 min. The samples were incubated with primary antibodies at 4°C overnight and washed three times with phosphate-buffered saline (PBS). Next, the samples were stained with fluorescent secondary antibodies for 1 h at 37°C. Primary antibodies against the following were used: vimentin (10366-1-AP, 1 : 200, Proteintech), E-cadherin (20874-1-AP, 1 : 200, Proteintech), N-cadherin (22018-1-AP, 1 : 200, Proteintech), SOX2 (ab79351, 1 : 100, Abcam), and Slug (#9585, 1 : 1000, CST). The following secondary antibodies were used: FITC-labelled goat anti-rabbit IgG (F9887, 1 : 500, Sigma) and DyLight 594-labelled goat anti-mouse IgG (ab96881, 1 : 500, Abcam). The nuclei were then counterstained with DAPI (Abcam) before imaging. IF images were captured using fluorescence microscopy (Leica Microsystems, Germany).

Hypoxia-treated cells (4 × 10⁶) were cross-linked by 1% formaldehyde treatment. Glycine was used to stop the cross-linking. Chromatin was sonicated and sheared into small fragments following the instructions of the EZ-ChIP™ kit (17-371, Millipore, Billerica, MA). One microgram of anti-RNA polymerase (05-623, Millipore, Billerica, MA), 1 μg of IgG (ab96881, Abcam), or 1 μg of anti-SOX2 antibody (ab79351, Abcam) was added to pull down the target protein. Target protein-bound DNA was purified and subjected to PCR amplification of an ~200 bp fragment of the β-catenin promoter using the following primer sequences: FW: 5′-GCCGAGTGGAAACTTTTGTCG-3′, BW: 5′-GGCAGCGTGTACTTATCCTTCT-3′.