Tumor tissues and the 3D gel were fixed with 4% paraformaldehyde for 72 h, sectioned, and subjected to sodium citrate antigen retrieval. LN229 or T98G cells were seeded on glass cover-slips in 6-well plates. The tissue and 3D gel sections and glass cover-slips with cells were fixed with 3% PFA/PBS for 20 min at 4 °C). The cells on glass cover slips were permeabilized with 0.1% Triton X-100/PBS for 10 min at room temperature. After washing with PBS three times, tissue sections and glass coverslips with cells were blocked with 5% goat serum in PBS for 30 min. Next, the sections and glass cover-slips were incubated with primary anti-p-PI3KTyr607 antibody (ab182651, Abcam, Cambridge, UK) and anti-p-AKT1S473 antibody (ab8932, Abcam, Cambridge, UK) for overnight at 4 °C, followed by incubation with goat anti-rabbit Alexa Fluor® 488 or donkey anti-rabbit Alexa Fluor® 594 secondary antibodies (Abcam, Cambridge, UK). Nuclei were stained with DAPI (Solarbio, Beijing, China) at room temperature for 5 min. Subsequently, photographs were acquired by a laser scanning confocal microscope (Olympus imaging Inc., Tokyo, Japan). The relative protein expression was analyzed by the fluorescence intensity. The mean fluorescence intensity (MFI) of a field (at least 50 tumor cells in each field) was calculated by image J software. A mean of 20 fields MFI was calculated as the MFI of the sample. A total of 10 samples in each group were used for the significant difference analysis.

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