Goat anti mouse igg

Thylakoid proteins were extracted as described above and separated by SDS–PAGE. Proteins were then transferred to nitrocellulose membranes, blocked with Tween tris-buffered saline (TTBS) buffer (20 mM Tris–HCl, pH 7.6, 0.137 M NaCl, and 0.1% Tween-20) containing 5% skimmed milk and incubated with the fused LTO1 protein carrying a poly-histidine tag at a concentration of 0.1 mg ml⁻¹ in TTBS buffer with 1% skimmed milk. The membrane was washed five times with TTBS buffer, probed with an anti-His-tag antibody (TA150088, Origene) and anti-HA-tag antibody (26D11, Abmart). The secondary antibody (goat anti-mouse IgG, Origene, ZB-2305) conjugated to HRP was used at a dilution of 1/10,000 for detection using the Pro-Light HRP Chemiluminescent Kit (Tiangen Biotech; Ouyang et al., 2011 (link)).

The liver tissues embedded in paraffin were sliced into 4–6 mm pieces, deparaffinized and serially dehydrated in ethanol. The sections were boiled for 3 minutes while immersed in EDTA-Tris (pH 9.0) for antigen retrieval and then treated with hydrogen peroxide for 30 minutes to inactivate the endogenous peroxidase. The sections were incubated overnight at 4 °C with primary antibodies, which included antibodies against α-SMA (1:400; Abcam, Cambridge, MA, USA), CD31 (1:2000; Abcam), CD34 (1:2500; Abcam) and von Willebrand factor (VWF) (1:200; Proteintech, Wuhan, China). The sections were subsequently incubated with the appropriate biotinylated secondary (goat anti-rabbit IgG and goat anti-mouse IgG; Origene Technologies, Beijing, China) for 30 minutes at room temperature, stained with Diaminobenzidine (DAB) and counterstained with hematoxylin. The positive areas were colored a brownish yellow. Image-Pro Plus software 6.0 was used for the immunohistochemical analysis.

The staining procedure was performed by standard streptavidin-horseradish peroxidase complex method. The slides were dried in an incubator at 65°C overnight, and following deparaffinization via 100% xylene for 15 min and hydration using 100% ethanol for 5 min, 95% ethanol for 2 min, 80% ethanol for 2 min and 75% ethanol for 2 min. Tissue sections were incubated with 3% hydrogen peroxide in methanol at room temperature for 30 min to block endogenous peroxidase activity. Briefly, the anti-PTTG (1:50 dilution; cat. no. 12575-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), anti-CD44v6 (pre-diluted; cat. no. 2M-0052; OriGene Technologies, Inc., Beijing, China), and anti-CD147 (CD147 Diagnostic kit; cat. no. CL001-01; Jiangsu Pacific-Meinuoke Biopharmaceutical Co., Ltd., Jiangsu, China) antibodies were applied and incubated at 4°C overnight, followed by washing with PBS. The sections were incubated with a secondary antibody (ready-to-use kit; cat. no. SP9000; goat anti-mouse IgG; OriGene Technologies, Inc.), for 30 min at room temperature. Signals were developed with 3,3-diaminobenzine substrate (1:200; cat no. ZLI-9018; OriGene Technologies, Inc.) for 2 min and counterstained with hematoxylin (ready-to-use; cat. no. BA-4041; Baso Diagnostic, Inc.) for 10 min at room temperature.

Total protein was extracted by ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology). The concentration of protein was detected using a BCA Protein Quantification kit (Beyotime Institute of Biotechnology). A total of 30 µg protein was loaded on a 10% SDS gel, resolved using SDS-PAGE and then transferred to a PVDF membrane (Invitrogen; Thermo Fisher Scientific, Inc.). After blocking with TBS with 0.1% Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.) containing 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies against FOXP3 (1:1,000; cat. no. ab20034; Abcam), E-cadherin (1:1,000; cat. no. ab40772; Abcam), Vimentin (1:1,000; cat. no. ab92547; Abcam) or GAPDH (1:1,000; cat. no. ab8245; Abcam) overnight at 4°C. The following day, the membranes were washed using TBST, and incubated with the HRP-conjugated goat anti-rabbit immunoglobulin G (IgG; 1:20,000; cat. no. ZB-2301; OriGene Technologies, Inc.) or goat anti-mouse IgG (1:20,000; cat. no. ZB-2305; OriGene Technologies, Inc.) secondary antibodies for 1 h at room temperature. Antigen-antibody complexes were detected using an enhanced chemiluminescence reagent (Cytiva). The relative expression of genes was analyzed using ImageJ (version 5.0; National Institutes of Health) and normalized to GAPDH.