Neutral resin

After conventional paraffin embedding, the human and mouse tumor tissues were cut into 4-μm histological sections, dewaxed with xylene, and hydrated with gradient density alcohol [16 (link)]. Antigen retrieval was conducted by boiling the sections for 10 min at 100°C in 10 mmol/L citric acid buffer (pH = 6.0). The sections were incubated with antibodies to E2F1 (1:500, sc-251, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), SESN3 (1:100, H00143686-M02, Abnova, Walnut, CA, USA), and ki-67 (1:1000, sc-23,900, Santa Cruz) at 4°C overnight. Next, the sections were re-probed with HRP-coupled anti-mouse secondary antibody (1:2000, ab205719, Abcam Inc., Cambridge, UK) for 60 min at room temperature. Peroxidase activity was observed with diaminobenzidine tetrahydroxyl chloride solution (Vector Laboratories, Inc., Burlingame, CA, USA), and the sections were counter-stained with hematoxylin. After gradient density alcohol dehydration, xylene dewaxing, neutral resin (Merck Millipore, Darmstadt, Germany) sealing, the staining results were observed under a microscope (Nikon Instruments Inc., Melville, NY, USA).

The miR-375 distribution in tissues was examined using an in-situ hybridization assay kit (Beyotime, Shanghai, China). The tissues were fixed in 4% paraformaldehyde for 0.5 h, and cultured with 0.25% acetic anhydride for 10 min before hybridization, followed by permeabilization with Triton X-100 for 10 min and proteinase K treatment to expose target nucleic acids. The sections were incubated with probe-free hybridization buffer for 120 min at ambient temperature to block nonspecific binding. Hybridization was conducted using a synthetic miR-375 probe. The sections were treated with 10 μL hybridization solution overnight at 42°C, washed with gradient concentrations of saline citrate (20 min each) at 37°C, and sealed with 3% BSA for 0.5 h at 37°C. Next, the sections were cultured with anti-digoxigenin anti-serum alkaline phosphatase complex overnight at 4°C. Finally, diaminobenzidine color development solution was added, and the section was placed in Tris-ethylenediamine tetraacetic acid solution for 10–30 min to terminate the reaction. After gradient density alcohol dehydration, xylene dewaxing, neutral resin (Merck Millipore) sealing, and the staining results were observed under a microscope (Nikon).