Tissue microarrays (TMAs) including the normal brain and low- and high-grade gliomas (total 91 patients) were made by PiNuoFei Bio Inc. Immunohistochemical (IHC) staining of TMAs were performed using the 3,3′-diaminobenzidine (DAB) staining method. After deparaffinization and antigen retrieval, TMAs were incubated with primary antibodies at 4°C overnight. Then, DAB (Origiene, Wuxi, China) and hematoxylin (Servicebio, Wuhan, China) were used to show positive expression sites and cell nucleus, respectively. The positive stain ratio of each LGALS in glioma patients was determined by IHC Profiler, an open-source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples (11 (link)). Primary antibodies used for IHC included anti-Gal-1 (Proteintech, Rosemont, USA 11858-1-AP, 1:1,000), anti-Gal-3 (Proteintech, 14979-1-AP, 1:1,000), anti-Gal-3BP (Proteintech, 10281-1-AP, 1:1,000), anti-Gal-8 (Invitrogen, Carlsbad, Amercian MA5-34693, 1:100; Santa Cruz, sc-377133, 1:50), anti-Gal-9 (Proteintech, 17938-1-AP, 1:1,000), anti-Arg1 (Proteintech, 16001-1-AP, 1:100), anti-CD206 (Abcam, ab64693, 1:100), anti-Iba1 (Abcam, ab5076, 1: 200), anti-SOX2 (Abcam, ab171380, 1:200), and anti-CD15 (CST, # 4744, 1:200).
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