Tissue embedder

Mouse forestomach carcinoma cell line (MFC) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai); aspirin was purchased from Sigma (St. Louis, MO, USA). RPMI-1640 medium (containing 15% fetal bovine serum, 100 µ/ml penicillin and 100 µ/ml streptomycin) were obtained from HyClone (Logan, UT, USA). Trypsin was obtained from Gibco (Grand Island, NY, USA). PBS was purchased from Shanghai Biological Engineering Company (Shanghai, China). MTT assay (5 mg/ml) was obtained from Sigma (St. Louis, MO, USA). Polyclonal rabbit anti-human E-cadherin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). FITC-labeled goat anti-rabbit IgG was from Beijing Zhong Shan Jinqiao Biological Technology Co., Ltd. (Beijing, China). Centrifuge was obtained from Thermo Fisher Scientific (Waltham, MA, USA) and syringes were from Bio-Rad (Berkeley, CA, USA). Inverted microscope was from Olympus (Tokyo Japan), Eppendorf tubes were obtained from Eppendorf (Hauppauge, NY, USA), super clean bench was from Thermo Fisher Scientific (Waltham, MA, USA), and cell counting chamber was from Shanghai Optical Instrument Factory (Shanghai, China). Paraffin slicing machine and tissue embedder were obtained from Leica (Mannheim, Germany) and ELISA plate reader was from Bio-Rad.

As described above, during post-surgery tumor recurrence follow-up, mice were imaged by CRI Maestro^TM (filter set: λ_Ex: 635 nm, λ_Em: 650-800 nm, filter: 675 nm) 4 h post i.v. injection of P-GFLG-Cy5 (10 µM Cy5-eq. concentration, 200 µL). Tissues that showed an increased Cy5 signal were suspected as recurrent or metastatic cancerous tissues, and were then excised and stained for H&E for further histo-pathological analysis. The suspected cancerous tissues were embedded in paraffin or OCT. Prior to OCT embedding, excised tissues were incubated with 4% PFA in PBS for 4 h followed by incubation in PBS for 12 h and 1 M sucrose (BioLab) overnight. For paraffin-embedded fixation, tissues were kept for 48 h in 4% PFA in PBS, then processed using a Tissue Processor (Leica) for 10 h and then embedded in paraffin using a Leica Tissue Embedder. 5 µm sections were deparaffinized, rehydrated, and stained by hematoxylin and eosin (H&E). Excised sternum tissues were fixed in 4% PFA and decalcified for 14 days in 14% ethylenediaminetetraacetic acid (EDTA), embedded in OCT and snap-frozen in liquid nitrogen prior to cryosectioning. 10 μm sections were obtained and stained by H&E. Sequential sections obtained from IGS were mounted with ProLong® Gold antifade reagent with DAPI and examined by confocal microscopy to evaluate Cy5 internalization into cells.

The LN28 glioblastoma cell line was purchased from the Chinese Academy of Sciences Cell Bank. The following experimental instruments were used: Micropipette tip (Rainin Instrument LLC, Oakland, CA, USA), optical microscope (Olympus Corp., Tokyo, Japan), polymerase chain reaction (PCR) TC-XP (Bioer Technology Co., Ltd., Hangzhou, China), constant temperature incubator (Changzhou Huapuda Instrument Co. Ltd., Changzhou, China), paraffin slicing machine (Leica, Mannheim, Germany) and tissue embedder (Leica). TRIzol reagent, RNase-free, reverse transcription (RT)-PCR primers, RT kit, quantitative PCR (qPCR) kit, Express SYBR-GreenER miR qPCR kits, NCode VILO miR cDNA (Invitrogen Life Technologies, Carlsbad, CA, USA) and the miR extraction kit (Roche Diagnostics GmbH, Mannheim, Germany) were employed. The rabbit polyclonal primary antibodies p-JAK2 (Cat. no. GTX101132, 1:500) and p-STAT3 (Cat. no. GTX110587, 1:500) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA.