Hematoxylin

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).

The femur samples were fixed with 4% PFA for 2 days and then embedded in paraffin after two weeks of decalcification with 10% EDTA treatment. Serial 5 μm sections of the femur were made using a microtome, and the femur sections were stained with immunohistochemical staining. Briefly, femur sections were processed for antigen retrieval for 15 min and blocked in 3% BSA for 30 min. The sections were then incubated overnight with primary antibody against osteocalcin (1:2000, Servicebio, China) at 4 °C. After three washes with PBS, the sections were incubated with HRP-conjugated anti-rabbit IgG (1:400, Servicebio, China) for 50 min. A 3N-diaminobenzidine tetrahydrochloride (DAB) kit (Servicebio, China) was used to detect immunoactivity, followed by counterstaining with hematoxylin (Servicebio, China). The sections were examined under a fluorescence microscope (Nikon, Japan).

Dewaxed sections were incubated with hematoxylin (Servicebio) and eosin (Servicebio); dehydrated with xylene, anhydrous alcohol, and 75% ethanol; and sealed with neutral gum (Servicebio). The cytoplasm and nucleus of all the cells turned to red and blue, respectively. The slices were scanned (Aperio AT2, Leica), and osteoclasts and tooth resorption lacuna were observed at the pressure side of the upper-left first molars.

A previously described peroxidase-based immunohistochemistry protocol was used to detect FHL2 expression in paraffin-embedded ovarian tissues [36 (link)]. Anti-FHL2 antibody (Proteintech, Wuhan, China) was used at 1:200. Subsequently, the sections were incubated with biotinylated secondary antibody (1:2000) (Boster Biotechnology, Wuhan, China) and avidin-biotin-peroxidase (Boster Biotechnology, Wuhan, China) before being exposed to diaminobenzidine (Servicebio, Wuhan, China, Wuhan, China) and counterstained with hematoxylin (Servicebio, Wuhan, China).

The minipigs were sacrificed 4 and 12 weeks after the operation, respectively, and the full-thickness vaginal graft/tissue explants were harvested and embedded in paraffin, then sectioned into 4 µm slices. H&E staining and Masson’s trichrome staining were performed according to the manufacturer’s instructions (Servicebio, China). Immunohistochemical staining was also performed with various primary antibodies, including CK14 (1:400; Servicebio, China), α-actin (1:200; Servicebio, China), CD31 (1:800; Servicebio, China), and PCNA (1:400; Servicebio, China) diluted in antibody diluent at 4 ^∘C overnight. After that, biotinylated secondary antibody (ZSGB-Bio, China) was applied for 1 h at room temperature and the samples were stained with 3,3-diaminobenzidine (DAB). The coloration was stopped with distilled water, and the nuclei were counterstained with hematoxylin (Servicebio, China). These slides were then examined using a light microscope (ZEISS, Germany).

For histological analysis, the fractured tibia was decalcified, paraffin was embedded, and the tibia was cut into 10 μm sections. Paraffin sections were rehydrated and stained with hematoxylin (Servicebio, Wuhan, China) for 1 min. After rinsing with water, the sections were stained with 0.2% Fast Green solution (Sigma Aldrich, USA), then incubated in 1% acetic acid for 12 s. Next, the sections were stained with 0.1% Safranin O solution (Sigma Aldrich, USA) for 3 min. The slides were washed, dehydrated, and mounted with resin mounting solution before taking images.