Ab181053

Coronal serial sections (3.5 µm thickness) were affixed to adhesive slides and stained with hematoxylin and eosin. Immunofluorescence staining was performed in accordance with standard protocols. Briefly, conventional dewaxing methods were performed; slides were then incubated in citrate buffer (pH = 6.0, PN5360, G-CLONE, Beijing, China) that was subjected to microwave heating for antigen retrieval. Subsequently, endogenous peroxidases were inactivated by incubation in 3% H₂O₂–methanol at room temperature for 10 min. Slides were then blocked with goat serum sealant (KGSP03, Keygen, Jiangsu, China) at room temperature for 20 min. Immunofluorescence staining was performed with a primary antibody against G-CSF (1:50 dilution, ab181053, Abcam, Cambridge, UK) via incubation in a humidified environment for 2 h at 37℃. Secondary antibody detection was performed with goat anti-rabbit fluorescein isothiocyanate (FITC) (1:50 dilution, 111-095-003, Jackson ImmunoResearch, West Grove, PA, USA), via incubation in darkness at 37℃ for 1 h. Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (KGA215, Keygen) was used for nuclear staining (5 min incubation at room temperature). Imaging was performed with a confocal microscope (BX43, Olympus, Tokyo, Japan).

KG1a cells were lysed by the non-denaturing lysis buffer. Next, the supernatant of cell lysis was pre-cleaned with protein A/G magnetic beads (Thermo Fisher Scientific) for 2 h at 4 °C. Subsequently, about 300 μg of protein were incubated with 1μg G-CSF antibody (#ab181053, Abcam) or EP300 antibody (#ab275378, Abcam) and 25 microliters of protein A/G magnetic beads for immunoprecipitation at 4 °C overnight. Following the incubation with antibody and protein A/G magnetic beads, protein A/G magnetic beads were collected using magnetic separation device (Thermo Fisher Scientific), and precipitated complexes were cleansed by washing buffer (Thermo Fisher Scientific). Finally, bound proteins were analyzed by WB using anti-ELANE (1:500, # bs-6982R, Bioss) or anti-TPO (1:500, # ab196026, Abcam). Rabbit IgG was used for negative control.

Crude cellular proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% non-fat milk and probed with primary antibodies (1:1,000 dilution) against CSF3 (Abcam, ab181053) overnight at 4°C. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1.5 h and immunolabeling detected using a Bio-Rad imaging system.