Gal 3

Cells were fixed with 4% polyformaldehyde for 20 min, blocked with 5% BSA in PBS and 0.2% Triton X-100 for 30 min. Cells were incubated with Gal-3 (1:100, Cell Signaling Technology) at 4°C overnight. Cells were incubated with goat anti-rabbit IgG-CFL 555 (1:100, Santa Cruz Biotechnology) at 37°C for 2 h after washing with PBST for 15 min and subsequently stained with DAPI in dark room for 15 min and washed with PBST for 15 min. Cells were analyzed using the Olympus BX51 fluorescence microscope.

All fresh tissue samples were fixed in 4% formalin buffer, embedded in paraffin, sectioned, and then deparaffinized. Antigen retrieval was performed using the wet autoclaving method in the presence of citrate buffer (pH 6.0). After blocking, specimens were incubated overnight at 4 with the following primary antibodies: NLRP3, ASC, Caspase-1 (ABclonal, USA; 1:700, 1:80, 1:30), and Gal-3 (Cell Signalling Technology, USA; 1:200). Next, the sections were incubated with anti-rabbit/mouse-HRP (ZSGB-BIO, China) and 10% normal goat serum at room temperature for 30 min. The DAB REAL EnVision Detection System (Agilent Technologies, USA) was used for color development. The slides were counterstained with modified Harris hematoxylin (Thermo Fisher Scientific, USA). The excess dye solution was washed away and the slides were then mounted using neutral gum mounting film. The sections incubated with phosphate-buffered saline solution were used as negative controls. Images were captured using a digital camera mounted on a light microscope (Leica DM2500 LED). The positive areas of NLRP3, ASC, Caspase-1, and Gal-3 were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). We used the IHC profiler plug-in to automatically score the stained samples [19 (link)]. All assessments were performed in a blinded manner.

For immunohistochemical staining of formalin-fixed paraffin-embedded tissues, antigen retrieval was performed in citrate buffer at pH 6.0 and microwave for 15 min. Sections were blocked, then incubated overnight at 4 °C with primary antibody Gal-3 (Cell signaling), pAKT (Cell Signaling), AKT (Cell Signaling), Ki67 (Chemicon), and CD31 (Abcam) followed by biotin-conjugated anti-rabbit IgG and an avidin–biotin peroxidase detection system with 3,3′-diaminobenzidine substrate (Vector), then counterstained with hematoxylin (Sigma). Immunofluorescence of formalin-fixed paraffin-embedded GSCs was performed as reported^{55 (link)}. The following primary antibodies against human antigens were used: rabbit anti-Gal-3 (Cell signaling) and mouse anti-RAB10 (Santa Cruz). The following fluorochrome-labeled secondary antibodies were used: Alexa Fluor (555 or 488)-labeled goat or donkey anti-mouse, or anti-rabbit antibodies. A Nikon Eclipse C1 Confocal microscope, as well as a Nikon Eclipse TE2000-E, were used for imaging.

Antibodies used in this study were from the following sources: LC3B (#2775, rabbit, WB: 1:1,000), FIP200 (#12436, rabbit, WB: 1:1,000), ATG7 (#8558, rabbit, WB: 1:1,000), ATG9A (#13509, rabbit, WB: 1:1,000), ATG16L1 (#8089, rabbit, WB: 1:1,000), and Gal3 (#87985, rabbit, IF: 1:400) were purchased from Cell Signaling Technology. Anti-beta-actin antibody (A2228, mouse, WB: 1:10,000) was purchased from Sigma-Aldrich. LC3 (PM036, rabbit, IF: 1:500) antibody was purchased from MBL International. ALIX antibody (634502, mouse, IF: 1:200) was purchased from BioLegend. IST1 (51002-1-AP, rabbit, IF: 1:100) and CHMP2A (10477-1-AP, rabbit, IF: 1:199) antibodies were purchased from proteintech. Gal8 antibody (NBP2-75501, rabbit, IF: 1:400) was purchased from Novus Biologicals. Goat anti-rabbit-HRP (Cat# 31460, WB: 1:10,000) and goat anti-mouse-HRP (Cat# 31430, WB: 1:10,000) antibodies were purchased from Thermo Fisher Scientific. Alexa Fluor 488/568/647-conjugated secondary antibodies for immunofluorescence were purchased from Thermo Fisher Scientific.
Reagents used in this study were from the following sources: Filipin (F9765) from Sigma-Aldrich; Bafilomycin A1 (BML-CM110-0100) from Enzo.