After routinely dewaxing and hydration, sections proceed to be antigen repaired with TE buffer at 98 °C. Each section was blocked with 3% H2O2. Each section was incubated with anti-Gli1 (Abcam), anti-CD44 (Abcam), anti-LSD1 (Sigma), anti-Sox2 (R&D), anti-Sox9 (Abnova), anti-LGR5 (Abcam), in primary antibody dilution buffer for 1 h at ambient temperature (AT). Then anti-mouse/rabbit antibody were used to incubated with tissue samples for 30 min at AT. Lastly, chromogenic agent 3, 3′-diaminobenzidine (Dako) was used to stain tissue samples.
The double immunostaining procedure was executed in the same section, the first step was to use anti-Gli1 antibody staining with 3,3′-diaminobenzidine, the second step was to use anti-CD105 antibody (Abcam) staining with AEC.
Two pathologists (WB Qi & YH Xuan) assessed the immunohistochemical results and the staining results were assessed according to previous study [9 (link)].
The double immunostaining procedure was executed in the same section, the first step was to use anti-Gli1 antibody staining with 3,3′-diaminobenzidine, the second step was to use anti-CD105 antibody (Abcam) staining with AEC.
Two pathologists (WB Qi & YH Xuan) assessed the immunohistochemical results and the staining results were assessed according to previous study [9 (link)].
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