Dimethylbenzene

As previously described [31 (link)], the CCA slides (n = 65) were baked at 60°C for 2 hours, then deparaffifinized and hydrated by dimethylbenzene(1330-20-7, Sigma-Aldrich) and ethanol (64-17-5, Sigma-Aldrich). Antigens were then repaired in citrate buffer (C9999, Sigma-Aldrich). Endogenous peroxidase was blocked with 3% H₂O₂ and then slides were blocked with bovine serum albumin (BSA) (A1595, Sigma). Next, the slides were incubated overnight along with the indicated primary antibodies. The next day, the slides were incubated together with the secondary antibodies. Samples were stained by dimethylbenzene (521116, Sigma-Aldrich) and hematoxylin (51275, Sigma-Aldrich), then sealed with neutral gum. All the tissue samples were scanned under a fluorescent microscope. IHC and protein expression scoring were described in previous reports [32 (link)].

As previous described (49 (link)), FFPE slides baked at 60°C for 2 hours before being deparaffifinized and hydrated by dimethylbenzene (Sigma-Aldrich, 1330-20-7) and ethanol (Sigma-Aldrich, 64-17-5). For IHC, antigens were then repaired in citrate buffer (Sigma-Aldrich, C9999). Endogenous peroxidase was blocked with 3% H₂O₂, and then slides were blocked with bovine serum albumin (BSA) (A1595, MilliporeSigma). Next, the slides were incubated overnight along with the indicated primary antibodies. The next day, the slides were incubated together with the secondary antibodies. Samples were stained by dimethylbenzene (521116, Sigma-Aldrich) and hematoxylin (51275, Sigma-Aldrich); they were then sealed with neutral gum. TUNEL assay was conducted by following the TUNEL assay kit (MilliporeSigma, C10617). Slides were deparaffinized and permeabilized with 4% paraformaldehyde for 15 minutes at 37°C. TdT reaction and Click-iT Plus reaction were conducted according to the protocol. A fluorescence microscope with 495 nM excitation was used to detect TUNEL, and 350 nM was used to detect DAPI. Expression quantitation were conducted by ImageJ (version1.53; NIH).