β catenin d10a8 xp

According to the manufacturer's instructions, paraffin-embedded tissue samples from consenting patients were incubated overnight using the following primary antibodies (1:50-100): SHMT2 (12762, Cell Signaling Technology), β-catenin (D10A8) XP (8480, Cell Signaling Technology). The IHC images were captured by a microscope (Olympus, USA). To get the relative expression (%) of the target protein, the measurement parameters collected by Image J included integral optical density (IOD), area and average optical density (AOD). The AOD represented the levels of specific protein per unit area.

CRC cells seeded on coverslips (Sarstedt Inc. TC coverslip 13 mm ST/CS200, Fisher Scientific) were fixed with 4% PFA in PBS for 20 min at rt. After washing three times with 0.2% BSA in Dulbecco's phosphate-buffered saline (DPBS), the cells were permeabilized with 0.1% Triton X-100 in PBS for 7 min. The permeabilized cells were blocked with DPBS containing 0.2% bovine serum albumin (BSA) for 30 min and then incubated with primary antibody (1:100-2000) overnight at 4°C, followed by Alexa-conjugated secondary antibody (1:200) incubation for 1 h at rt. The coverslips were mounted in DPBS supplemented with DABCO. All IF images were acquired with a laser scanning confocal microscope (Leica SP8 X, Leica) equipped with LAS X software, using a 63x3 1.3 NA oil objective. The following antibodies were used: mSHMT (sc-390641, Santa Cruz Biotechnology), TOM20 (11802-1-AP, Proteintech), β-catenin (D10A8) XP (8480, Cell Signaling Technology), E-cadherin (A11509, ABclonal), vimentin (10366-1-AP, Proteintech), goat anti-rabbit IgG (H+L) cross-adsorbed Alexa Fluor 488 (A-11008, Invitrogen), and goat anti-mouse IgG (H+L) cross-adsorbed Alexa Fluor 568 (A-11031, Invitrogen).