Ctla 4 antibody

Immunohistochemistry (IHC) was performed as previously reported [23 (link)]. Briefly, antigen retrieval was carried out in EDTA solution at 100°C, pH 9.0 for 10 minutes. After blocking of the nonspecific antigens, the tissue section was incubated with CTLA-4 antibody (Abcam, Cambridge, UK) overnight at 4°C. For antibody visualization, liquid 3,30-diaminbenzidine (DAKO) was used.
Tumor cells and interstitial lymphocytes were stained with CTLA-4. The expression levels of CTLA-4 were defined as follows: “−” (negative), “+” (weakly positive), “++” (moderately positive), and “+++” (strongly positive). Tissue sections with “++” and “+++” were defined as a high expression of CTLA-4 [24 (link)]. The IHC analysis was performed by two independent pathologists blinded to the clinical information.

Diethylnitrosamine (DEN) was purchased from Sigma (N0258, Sigma-Aldrich, USA). Carbon tetrachloride was purchased from Hao-Sen Chemical Co., Ltd. (Shanghai, China). Ethanol (55%) was purchased from Niu Lan Shan Co., Ltd. (Beijing, China). The TRI Reagent was purchased from Sigma (T9424; Sigma-Aldrich, USA). The Revert Aid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher (K1621, Thermo Fisher; U.S.A). HieffTM qPCR SYBR® Green Master Mix was purchased from Yeasen Biotech Co., Ltd. (Cat. No. 11203, Shanghai, China). Anti-pan-AKT antibody (#ab8805, Abcam, Cambridge, U.K.), anti-p-AKT (ser473) antibody (#ab126433, Abcam, Cambridge, U.K.), Anti-AFP primary antibody (cat# ab46799, Abcam, Cambridge, U.K.), β-actin antibody (cat# ab8227, Abcam, Cambridge, U.K.), anti-IL-10 primary antibody (cat#ab189392, Abcam, Cambridge, U.K.), and CTLA-4 antibody (cat#ab134090, Abcam, Cambridge, U.K.) were purchased from Abcam (UK). CD4 (cat#25229), CD8α (cat#98941), and PD-1 (cat#84651) antibodies were purchased from Cell Signaling Technology (M.A. USA).