Xenograft tumor in nude mice and immunohistochemistry (IHC)

All animal experiments were conducted in accordance with animal protocols approved by the Institutional Animal Use and Care Committee of the affiliated Union Hospital of Huazhong University of Science and Technology. Six male nude mice were randomly divided into two groups and a total of 1 × 10⁶ 786-O cells with sh-LINC00160 or NC were injected into the flank of mice subcutaneously. Tumor size was measured every 3 days. Tumor volumes were calculated by (D × d²)/2. The performer was blinded to the group allocation during the experiment. The mice were euthanized and the tumors were harvested after 30 days. Then, six xenograft tumor were fixed in formalin, dehydrated, and embedded. The tissue sections were incubated with primary rabbit Ki67 (1:100; A11005; ABclonal Biotech Co., Ltd) and then incubated with secondary antibody for 2h at room temperature.