Alexafluor 546 donkey anti rabbit a10040

Primary CRC cells were seeded and grown in 12-well cultivation chambers with removable microscopy glass slides (ibidi, Martinsried, Germany); cancer spheroids obtained by the hanging drop assay were also sedi-mented in the same chamber. Immunofluorescence analyses were performed as previously described by Di Maio et al (31 (link)). Briefly, following fixation in 4% paraformaldehyde in PBS for 10 min, the cells were permeabilized in 0.1% Triton X-100 in PBS for 30-120 min, and then blocked in 10% bovine serum albumin for 30 min. The cells were incubated with primary antibodies (Table I) overnight, and then with secondary antibodies (Alexa Fluor 546 donkey anti-rabbit, A10040; Alexa Fluor 488 donkey anti-mouse, A21202; Thermo Fisher Scientific) for 1 h, and then with DAPI (Sigma-Aldrich) for 30 min at room temperature to label the nuclei. Negative controls without primary antibodies were also included, and these exhibited no staining. Following the indicated treatments, coverslips were mounted on glass slides and examined under a fluorescence confocal microscope (Zeiss LSM 700, Carl Zeiss, Oberkochen, Germany).