Dylight 549

Cells were seeded at a density of 2 × 10⁴/well on 4-well Lab-Teck II chamber slides (Nunc, Naperville, IL, USA). After indicated treatment, cells were washed twice with ice-cold PBS and fixed immediately with 4% paraformaldehyde in PBS for 15 minutes at room temperature. After two washes with PBS, cells were permeabilized with 0.25% Triton X-100 for 10 minutes at room temperature. Then cells were blocked with 1% bovine serum albumin (BSA) in PBS/Tween 20 for 30 minutes at room temperature and further incubated overnight with the primary antibodies against TrxR1 (Abcam, 1:300), CD44-PE (eBioscience, 1:160), E-Cadherin (BioLegend, 1:300) or N-Cadherin (Biolegend, 1:300) diluted in 1% BSA in PBS/Tween 20 in a humidified chamber at 4 °C. After three rinses with PBS, secondary antibodies conjugated with Dylight 488, Dylight 649 or Dylight 549 (Abbkine, 1:300) diluted in 1% BSA in PBS/Tween 20 (1:200) was added and incubated for 1 hour at room temperature in dark followed by three washes with PBS. Subsequently, nucleus were counterstained with 4′,6′-diamino-2-phenylindole (DAPI) for 10 minutes. Images were taken with a laser scanning confocal microscope (Nikon A1, Japan) and analyzed by NIS-Elements Viewer 4.20 software.

Forty percent confluent cultured HeLa cells grown on coverslips were serum-deprived for overnight and treated with CoCl₂ for 12 h. Then coverslips were fixed using 4% paraformaldehyde, permeated with 0.1% Triton X-100, and incubated with blocking buffer (10% goat serum in PBS) at room temperature for 1 h. After PBS wash, coverslips were incubated with VEGF-A (Abcam) or HIF-1α (Novus) antibody overnight at 4 °C, and then incubated with Dylight 549 (Abbkine, USA) for 1 h at room temperature. 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime, China) was used for nuclear staining. Nikon A1⁺ R Real-Time Full-Spectrum Double Sweep Laser Scanning Confocal Microscope supported by quantification and image processing software NIS-Elements 4.3 (Nikon, Japan) was used for experiment evaluation.
Fresh resected tumor tissues were embedded in O.C.T. compound (Sakura, USA) and sliced into consecutive sections with a thickness of 10 μm utilizing a vibrating microtome (Leica CM1950, Nussloch, Germany). The slices were incubated with VEGF-A (Abcam) or Ki-67 (Abcam) antibody overnight at 4 °C

Cells grown on glass coverslips and frozen sections of brain tissue were lightly fixed in 4% paraformaldehyde, blocked with 3% FBS/0.01% Triton X-100 in PBS, and incubated at 4 °C overnight with the following primary antibodies: anti-DJ-1 (1:50, Abcam, ab76008), anti-Nrf2 (1:50, Abcam, ab31163), and anti-GFAP (1:200, ABclonal, A10871). The next day, cells or sections were incubated with secondary antibodies at room temperature. Secondary antibodies were tagged with either DyLight 488 (anti-mouse, Abbkine, green, A23210) at 1:200 or DyLight 549 (anti-rabbit, Abbkine, red, A23320) at 1:200. Coverslips and sections were mounted with Vectashield containing DAPI. Sections and cells were imaged with a fluorescence microscope or a laser scanning confocal microscope.