Immunohistochemistry Analysis of NLRP3 and IL-1β

IHC was performed described previously [44 (link)]. Wax blocks containing tissue blocks were sectioned. The reaction was fulfilled for 30 min with a blocking solution containing 10% calf serum (Gicbo, Life Technologies, Carlsbad, CA). The primary antibodies (anti-Ki-67 [ab271811, 1 : 100, Abcam], anti-NLRP3 [ab214185, 1 : 100, Abcam], anti-caspase-1 [ab62698, 1 : 100, Abcam], and anti-IL-1β [ab283818, 1 : 500]) were added overnight at 4 °C, and PBS (Hyclone, Logan, USA) was used as a negative control instead of primary antibodies. The secondary antibodies were added dropwise with biotin-labeled IgG (ab205719, 1 : 1000, Abcam) and reacted at 37 °C for 1 h. Finally, DAB (Dako, Denmark) was used for color development and hematoxylin contrast staining and sealed with gelatin. The scoring criteria for immunohistochemistry were done by the physicians of the pathology department of our hospital.