Ap1802a

The IHC staining was performed on 4 μm paraffin sections. The sections were dewaxed, hydrated, and placed at 4 °C overnight. For antibodies against CD133 (AP1802a, Abgent, San Diego, CA, USA), P62 (ab56416, Abcam, Cambridge, MA, USA) and LC3II (AP1802a, Abgent, San Diego, CA, USA), the standard avidin-biotin complex (ABC) procedures were employed. After the sections were returned to room temperature, biotinylated secondary antibodies and horseradish-labeled streptavidin were added. The samples were then incubated in an oven at 37 °C Subsequently, DAB color development, hematoxylin counterstaining, gradient alcohol dehydration, and xylene transparent were carried out. All samples were sealed with neutral gum afterwards.

Immunohistochemical staining was performed on 4-μm-thick paraffin sections. The sections were dewaxed hydrated and placed at 4 °C overnight. For antibodies against CD133 (AP1802a, Abgent, San Diego, CA, USA), P62 (ab56416, Abcam, Cambridge, MA, USA), and LC3II (AP1802a, Abgent), the standard avidin–biotin complex procedures were employed. After the sections were returned to room temperature, biotinylated secondary antibodies and horseradish-labeled streptavidin were added. The samples were then incubated in an oven at 37 °C. Subsequently, DAB color development, hematoxylin counterstaining, gradient alcohol dehydration, and xylene transparent were conducted. All samples were sealed with neutral gum afterwards. Human brain tissues: the ethics statements in this study were approved by the Institutional Review Board of Kaohsiung Medical University Hospital (No. KMUHIRB-F(I)-20200024). Informed consent was obtained from all subjects involved in the study.

Protein expression was detected by immunoblot analysis. Cell lysates with equal protein content were prepared in SDS sample buffer, separated on NativePAGE Novexw Bis–Tris 4–16% gel for BN-PAGE analysis (Invitrogen; Thermo Fisher Scientific, Inc.) followed by transferring to polyvinylidene fluoride membranes. Proteins on the membrane were detected with specific primary antibodies and HRP-conjugated secondary antibodies. The following antibodies were used: P62 (1:2000; ab56416, Abcam, Cambridge, MA, USA), β-actin (1:1000; ab3280, Abcam, Cambridge, MA, USA), LC3II (1:1000; AP1802a, Abgent, San Diego, CA, USA), Fzd 1 (1:1000; GTX108181, GeneTex, Irvine, CA, USA), GAPDH (1:10000; GTX100283, GeneTex, Irvine, CA, USA), GSK3β (1:1000; BD610202, BD Biosciences, San Jose, CA), GSK3β Ser9 (1:1000; #9323s, Cell Signaling Technology, Inc.), β-catenin (1:1000; #9562s, Cell Signaling Technology, Inc.), β-catenin Ser33/37/41 (1:1000; #9561s, Cell Signaling Technology, Inc.), CD133 (1:1000; #5860s, Cell Signaling Technology, Inc.), CD44 (1:1000; ab157107, Abcam, Cambridge, MA, USA), Nestin (1:1000; ABD69, Millipore Sigma, Burlington, MA), SOX-2 (1:1000; ab97959, Abcam, Cambridge, MA, USA), and β-actin (1:1000; sc47778, Santa Cruz Biotechnology, Santa Cruz, CA). The signal of each target protein was visualized by incubation with ECL Reagent and exposure to X-ray film.