Cells were fixed and permeabilized with 0.1% Triton X‐100. The cells were blocked with 5% bovine serum albumin (BSA) and then immunostained with rabbit anti‐PLK4 antibody (1:200; Abcam), mouse anti‐Ki67 antibody (1:200; Abcam), or mouse anti‐γ‐tubulin antibody (1:200; Sigma‐Aldrich, USA), followed by incubation with a goat anti‐rabbit or goat anti‐mouse secondary antibody (1:400; Invitrogen), phalloidin (1:400; Abcam), and 4',6‐diamidino‐2‐phenylindole (1:800; Sigma‐Aldrich). Samples were mounted with an anti‐fade reagent (Cell Signaling Technology, USA). Cell area analysis was performed using ImageJ software. The relative cell areas were normalized to NFs. The immunofluorescence staining of KFs and NFs was conducted on at least three biological replicates.
Tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. The sections were blocked with 5% BSA and incubated with a rabbit anti‐PLK4 antibody (1:200; Abcam). After sequential incubation with a biotinylated secondary antibody (Dako, Denmark) and an ABC‐alkaline phosphatase complex (Vector, USA), specific staining was revealed by using Fast Red (Dako). Immunohistochemistry staining of keloid and adjacent normal skin samples was conducted on at least three biological replicates.
Tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. The sections were blocked with 5% BSA and incubated with a rabbit anti‐PLK4 antibody (1:200; Abcam). After sequential incubation with a biotinylated secondary antibody (Dako, Denmark) and an ABC‐alkaline phosphatase complex (Vector, USA), specific staining was revealed by using Fast Red (Dako). Immunohistochemistry staining of keloid and adjacent normal skin samples was conducted on at least three biological replicates.