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Subsequently, OCT agent was washed off using PBS, and the samples were permeabilized with 0.25% Triton X-100 at room temperature for 20 min, blocked with 5% bovine serum albumin at room temperature for 1 h, and incubated with the primary antibodies: anti-ASCL4 (1:200, ABclonal, A6826, USA), anti-GPX4 (1:200, ABclonal, A13309, USA), anti-xCT (1:500, Affinity, DF12509, USA), anti-TfR1 (1:200, ABclonal, A5865, USA), and anti-ZO-1 (1:200, Abcam, ab68477, USA) at 4 C overnight