Tc coverslip 13 mm st cs200

Treated cells were seeded on coverslips (Sarstedt Inc. TC coverslip 13 mm ST/CS200, Fisher Scientific) were fixed with 4% PFA in PBS for 15 min at room temperature. Cells were blocked with 1% BSA dissolved in PBS for 1 h after permeabilized with 0.1% Triton X-100 in PBS for 7 min, then incubated with primary antibody anti-5-mC (28692, Cell Signaling Technology) overnight at 4 °C, followed by Fluor 488-conjugated (1:200) incubation for 1 h at room temperature. The tablets were sealed with an anti-fluorescence quencher containing DAPI (P0131, Beyotime). IF images were acquired with a laser scanning confocal microscope (Andor) equipped with Imairs Viewer software.

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).