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The tissues were rinsed with PBS and incubated with Alexa Fluor488 chicken anti-rabbit (1:1000, Life Technologies) for one hour at 37 C and then rinsed with PBS and incubated with Alexa Fluor488 goat anti-chicken antibody (1:1000, Life Technologies) and Alexa Fluor568 goat anti-mouse antibody (1:1000, Life Technologies) for another hour at 37 C

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The 3 targeting region was digested with Bam HI/ Xba I and cloned into Bam HI/ Xba I pL0001-5 gst -digested plasmid to create the final pbgst-ko plasmid

Specimens were taken from 3 areas by punch biopsy to clarify the diagnosis