Tissue tek optimal

After treatments, the tissues were transferred to Tissue-Tek optimal cutting temperature (O.C.T, Sakura Finetek, Tokyo, Japan) compound to snap freeze for immunohistology. IHC was done as previously described [32 (link),34 (link)]. Briefly, 10 µm tissue sections were air-dried on the slide glasses (Matsunami Glass, Osaka, Japan) for 40 min at RT, then washed with tris-buffered saline with 0.1% Tween20 (1× TBST) for 10 min. Tissues were blocked with 3% bovine serum albumin (BSA Gemini, Bio-Products, West Sacramento, CA, U.S.A.)/1× TBST for 1 h followed by overnight incubation with vimentin diluted at 1:300 (Abcam, Cat. No.: ab92547, Lot No.: GR3186827-13) and cytokeratin-18 diluted at 1:800 (Abcam, Cat. No: ab668, Lot No: GR3196069-6) in 3% BSA/1× TBST at 4°C. The next day, tissues were washed 3 times with 1× TBST for 10 min each, then treated with secondary antibodies diluted at 1:1000 (Alexa Fluor® 488, Abcam, Cat. No.: ab150073, Lot: GR269274-4 and Alexa Fluor™ 594, Invitrogen, Cat. No.: A11005, Lot: 2179228, respectively) in 3% BSA/1× TBST for 3 h at RT. Then, tissues were stained with NucBlue™ Fixed Cell ReadyProbes™ (DAPI, Invitrogen, Cat. No: R37606, Lot No: 2216969) for 5 min, and washed 3 times with 1× TBST for 10 min each. The images were taken with a Keyence microscope (Keyence Corp., Osaka, Japan).