H e staining kit

The animal experimental procedures were conducted in accordance with the guidelines of our institution. The procedures were approved by the animal ethic committee of our institution. Nude mice were obtained from SLAC (Shanghai, China) and maintained in a 12 h light/dark, specific pathogen free condition. The HuCCT1 cells were infected with scramble, lnc-PKD2-2-3 overexpression, and lnc-PKD-2-3 knockdown lentivirus (Genepharma, China) to generate scramble, oeLnc, and shLnc cells, respectively. The scramble, oeLnc, and shLnc cells were inoculated subcutaneously into the dorsal flanks of mice (N = 6 for each group) with an amount of 2.5 × 10⁶. The tumor sizes were measured every week for 4 weeks and calculated using the formula: volume = length × width²/2. The mice were sacrificed at 4 weeks after cell implantation. The tumors were harvested and weighed. Each tumor was split into two, and stored in −80°C or embedded in paraffin. The paraffin embedded tumors were cut into 4 μm sections for hematoxylin–eosin (HE), TdT-mediated dUTP-biotin nick end labeling (TUNEL), and immunohistochemistry (IHC) staining, with the involvement of HE staining kit (Sangon, China), TUNEL staining kit (Beyotime, China), and GPAM antibody (1:40) (Invitrogen, USA). The frozen tumors were lysed for RT-qPCR and western blot.

Dorsal mouse skin samples were fixed overnight with 4% paraformaldehyde in phosphate buffered saline (PBS) at 4 °C and then embedded in paraffin. Paraffin sections (4 μm) were mounted on silane-coated slides and stained with Hematoxylin&Eosin (H&E) using H&E staining kit (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China). The sections were examined, and images were recorded using a microscope and Image-Pro Express 5.1.1.14 Pathology Image Analysis System (Olympus Corporation, Tokyo, Japan) at 400× magnifications. The epidermal thickness was measured at 8 random sites randomly selected locations per slide using the image analysis program.

Paraffin-embedded sections of lymph nodes were subjected to xylene and gradient concentration of ethanol treatment and tumor cell infiltration in lymph node tissues was analyzed using the HE Staining Kit (E607318, Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China) according to the instructions of the kit. After dewaxing and rehydration, the sections were stained with hematoxylin for 5 min, treated with acetic acid, washed with ethanol, stained with eosin staining solution for 1 min, dehydrated with gradient ethanol, cleared with xylene, and sealed with neutral gum. The infiltration of tumor cells in the lymph nodes of nude mice was observed and confirmed by pathologists under a light microscope.