Ar9961

Immunohistochemistry was done using 4 µ thick sections of FPPE tissue. Immunohistochemical stains for GFAP clone EP672Y (Cell Marque 258R-16, lot 32653, 1:25), MN1 (Proteintech 24697-1-AP, lot 00021048, 1:40) and IGF2 (Abcam; ab9574, lots GR31975-61, and GR31975-64, 1:500) were performed on the Leica Bond III using Leica Epitope Retrieval 1 (Leica; AR9961) for GFAP, and Epitope Retrieval 2 (Leica; AR9640) for MN1 and IGF2. Retrieval was performed for 20 min, followed by 15 min primary antibody incubation, and Bond Polymer Refine Detection (Leica; DS9800). Hematoxylin was used as a counterstain. Positive IGF2 staining was defined as dark granular perinuclear cytoplasmic staining.

Immunofluorescence analysis was performed according to our previous study [25 (link)]. First, the tumor and/or major organ tissues were embedded in paraffin and cut into 2-μm sections using a slicer (Leica, Bensheim, Germany). 4T1 and HeLa cells cultured on glass coverslips were also treated with different preparations for 4 h. Then, the cells and tissue sections were fixed with 4% paraformaldehyde at 4 °C for 30 min and antigens were retrieved with an AR buffer (Leica, AR9961) and permeabilized with 0.1% Triton X-100 in PBS for 20 min. After blocking with 5% BSA at room temperature for at least 30 min, the samples were incubated with primary antibodies at 4 °C overnight. After washing three times with PBS, the samples were incubated with an indirect immunofluorescence secondary antibody at room temperature for 1 h, while cell nuclei were stained with DAPI for 5 min in the dark. Images were acquired with a confocal fluorescence microscope (ZEISS, LSM 980 with Airyscan 2, Oberkochen, Germany). For autophagy flux analysis, HeLa cells were transfected with mCherry-GFP-LC3B tandem reporter and imaged at 48 h post-transfection by confocal fluorescence microscopy.