Immunohistochemical Analysis of Transcription Factors

IHC was performed as described previously [80 (link)]. Paraffin-embedded clinical tissues were incubated with the following antibodies: anti-SOX9 (abcam, ab185966; 1:1000 dilution), anti-RUNX2 (abcam, ab192256; 1:1000 dilution), anti-TTF1 (abcam, ab76013; 1:250 dilution), anti-Ki67 (Proteintech, 27309-1-AP; 1:5000 dilution), anti-SOX4 (abcam, ab243041, 1:1000 dilution), anti-RUNX1 (abcam, ab240639, 1:2000 dilution), anti-SIX1 (abcam, ab252224, a:100 dilution), and anti-Clusterin (abcam, ab92548; 1:200 dilution). The immunostaining was reviewed and scored blindly. The scoring system for grading expression level was reported previously [81 (link)]. The score of each sample was multiplied by the grading of intensity and staining area.