Bovine serum albumin (bsa)

The distal colon tissues were fixed with 4% paraformaldehyde before embedding in paraffin, sectioned at a thickness of 4 um, dewaxed, and subjected to antigen repair with citric acid (VECTOR Laboratories, USA) at a high temperature, and then blocked with 5% bovine serum albumin (Absin, China) for 30 min. The dilution solution of the primary antibody was incubated at 4°C overnight, and the dilution ratios of the anti-Ly6C antibody, anti-F4/80 antibody, and anti-Ly6G antibody were 1:300. The antibodies were purchased from HUABIO (Zhejiang, China). Immunohistochemical secondary antibodies were incubated with enhanced HRP-labeled sheep anti-mouse/rabbit IgG polymer (ZSBB-Bio, CHINA) for 30 min at room temperature, followed by treatment for 10 min with DAB, counterstaining with hematoxylin, and finally sealing with neutral resin. ImageJ analysis was used to calculate the positive results of the images captured with the VS120 microscope (Olympus, Tokyo).

Cell proliferation was examined with a CCK8 assay, and the soft agar assay was performed as previously described.¹⁶ For the tumorsphere formation assay, 500 cells were seeded in 96‐well ultra‐low attachment plates (Corning Inc) with DMEM supplemented with 2% B‐27 supplement (Invitrogen), 10 ng/mL basic fibroblast growth factor (Novus), 20 ng/mL epidermal growth factor (PeproTech), 5 μg/mL insulin (Solarbio), and 0.4% bovine serum albumin (Absin). Colonies were counted after 7 d of incubation.

The GEM-RPC cells from vehicle and pralatrexate monotherapy treatment groups in 6-well plates were washed three times. The cells were then fixed with 4% paraformaldehyde (PFA) for 20 min, followed by 20 min of incubation with 0.5% TritonX-100 in PBS. Subsequently, the cells were incubated with blocking buffer (4% bovine serum albumin (BSA) (Absin, Shanghai, China) in PBS) for 1h at room temperature. All cells were washed with PBS three times after permeabilization and blocking. Primary antibodies were diluted at the suggested ratios and incubated at 4 °C overnight. Then, all cells were washed with PBST at room temperature three times. Appropriate secondary antibodies (#AS007) (ABclonal, Wuhan, China) were added and incubated for 1 h at room temperature in dark environment. Finally, the cells were treated with DAPI (MCE, China) for 5 min in a dark environment and washed with PBST at room temperature. The confocal microscope (OLYMPUS IX83-FV3000-OSR, Tokyo, Japan) were used to photograph pictures.

NBT (98%) and allopurinol (98%) were purchased from Aladdin Industrial Corp. (Shanghai, China). XOD and xanthine (≥99%) were purchased from Sigma-Aldrich Chemical Corp. (St. Louis, MO, USA). Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-2Na·2H₂O, ≥ 99.0%), dipotassium hydrogen phosphate trihydrate (K₂HPO₄·3H₂O, ≥ 99.0%), potassium dihydrogen phosphate (KH₂PO₄, ≥99.5%), and dextran (Mw 20,000) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glycerol (99%) was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Absin Bioscience Inc. (Shanghai, China). Trehalose (≥99%) was purchased from Sinozyme Biotechnology Co., Ltd. (Nanning, China). Ultrahigh-purity water was produced using a Milli-Q water purification system from Millipore (Milford, MA, USA). The 3D printing materials, namely, polycaprolactone, wax wire, and polylactic acid (PLA) filament, were purchased from Dongguan Top Cool Electronics Technology Co., Ltd. (Dongguan, China), Fuzhou Zhilei Electronic Technology Co., Ltd. (Fuzhou, China), and Hangzhou Shining 3D Technology Co., Ltd. (Hangzhou, China), respectively. Salvia miltiorrhiza (batch No. 170901) was purchased from Jiuzhou Drugstore (China Jo-Jo Drugstores Inc., Hangzhou, China).