Chromogenic substrate

Immunohistochemistry assay was performed according to our previously established protocols[14 (link),15 (link)]. The tissue slides were blocked with 3% BSA for 2 h at room temperature followed by overnight incubation at 4 °C with primary antibodies against MTDH (1:50) and RELA (1:50). The following day, the slides were incubated with SignalStain^® Boost IHC detection reagent (Cell Signaling Technology; Danvers, MA, United States) for 2 h at room temperature. After washing, chromogenic substrate (Thermo Fisher Scientific; Waltham, MA, United States) was applied to visualize the staining of the target proteins. Following counterstaining with hematoxylin, the sections were dehydrated and mounted using a coverslip.

To determine the concentration of pro-inflammatory cytokines in serum or the relative expressions of proteins inside lymphocytes, the serums or supernatants of lymphocyte lysates were serial diluted and 3 dilutions were loaded into each well of 96 well plates. Five dilutions of standard samples (50 µL, Shenzhen Jingmei Biological Engineering Co., Ltd., China) of desired proteins were loaded as control. The plates were then washed by TBS buffer, dried and then blocked by 3% BSA. Primary antibodies (100 µL) previously used in western blotting or antibodies of IFN-γ, IL-2, IL-4 and IL-10 (Abcam) were added into plates. After 1 h of incubation, the plates were washed and again incubated with 100 µL HRP conjugate secondary antibodies. The plates were then washed twice with TBS buffer and dried. Chromogenic substrates (100 µL, Thermo fisher scientific) were then added the plates and reacted for 30 min at room temperature in dark. Stop solution (100 µL, Thermo fisher scientific) were then added into each well, and the absorbance detected using Varioskan LUX Multimode Microplate Reader (Thermo fisher scientific) at the wave lengths of 450 nm and 550 nm. The absorbencies were normalized based on the data of standard samples. All of the experiments were repeated three times.