Gb23301

Protein was extracted from the cells were resolved by SDS-PAGE and then transferred to PVDF membranes (IPVH00010, Millipore), and then incubated with primary antibodies diluted in blocking buffer at 4 °C overnight. The following primary antibodies were used: Mouse Anti-β-Actin (HC201, TransGen Biotech, 1/2000), HRP conjugated Goat Anti-Mouse IgG (H + L) (GB23301, Servicebio, 1/2000), Rabbit Anti EMP3 (DF14661, Affinity, 1/1000), Rabbit Anti TWIST1(AF4009, Affinity, 1/1000), Rabbit Anti SNAI2 (PB9439, Boster, 1/1000), Rabbit Anti FOS (AF5354, Affinity, 1/1000), Mouse Anti-VIM (60330–1-Ig, Proteintech,1/10000), HRP conjugated Goat Anti-Rabbit IgG (H + L) (GB23303, Servicebio, 1/2000), HRP conjugated Goat Anti-Mouse IgG (H + L) (GB23301, Servicebio, 1/2000)was used. Finally, the antigen–antibody reaction was visualized by the enhanced Pierce ECL Western blotting substrate kit (Thermo Scientific/ Pierce, Rockford, IL, USA).

H1975 cells were treated with RIPA lysis buffer and protein concentration was measured using the BCA protein assay kit (E-BC-K318-M, Elabscience). After extraction, proteins were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, Massachusetts, USA). The 5% non-fat milk were used to block above membranes, and we incubated them with the Anti-SPC25 antibody produced in rabbit (1:1000, DF13747, Affinity Biosciences) or Mouse anti-β-actin (1:2000, HC201, TransGen Biotech) at 4°C overnight. The membranes were washed with TBST, then incubated with corresponding secondary antibodies for 1 h at room temperature, namely HRP conjugated Goat Anti-Rabbit IgG (H+L) (1:2000, GB23303, Servicebio) and HRP conjugated Goat Anti-Mouse IgG (H+L) (1:2000, GB23301, Servicebio). Protein levels were detected using an enhanced chemiluminescence (ECL) system.

The clinical samples were fixed in 10% neutral buffered formalin for 24 h. Paraffin was used for tissue embedding. Then tissue slides were well prepared and deparaffinized using dimethylbenzene, anhydrous ethanol, 85% ethanol, 75% ethanol, and distilled water orderly. The container with ethylene diamine tetraacetic acid (EDTA) antigen repair buffer (PH 9.0, Servicebio, G1203) for MYD88 or citric acid tissue antigen repair solution (PH 6.0, Servicebio, G1202) for CD68 and CD163 in the microwave oven was used correspondingly to repair the antigen of the slides using median fire to boiling for 8 min, keeping warm for 8 min and median-low fire for 7 min consecutively. Peroxidase was blocked using the 3% H₂O₂ for 25 min. Then we blocked the antigen using goat serum (Servicebio, G5001) for 30 min. We used MYD88 (1:20, CST, 4283S),CD68 (1:200, Servicebio, GB14043) and CD163 (1:600, Servicebio, GB13340) antibody overnight at 4°C to stain the slides, among which the two adjacent slides were stained with CD68 and CD163 separately. Then the slides were incubated with secondary antibodies (1:200, Servicebio, GB23303) for MYD88 and CD163 or secondary antibodies (1:200, Servicebio, GB23301) for CD68 50 min at room temperature. After adding DAB (DAKO, K5007) and hematoxylin (Servicebio, G1004) staining, slides were covered and observed by microscope (Grundium OCUS).