510 meta nlo confocal microscope

Duolink in situ PLA analysis was performed according to the manufacturer’s instructions (Olink Biosciences). Briefly, paraformaldehyde-fixed cells were washed with PBS, incubated for 10 min in 0.1% (volume/volume) Triton X-100, washed, and blocked with blocking solution. Primary antibodies, against ERK1/2 and Phospho-ERK1/2 antibodies, were applied, and the cells were incubated with PLUS and MINUS secondary PLA probes against rabbit and mouse IgGs. The incubation was followed by hybridization and ligation, and then amplification was performed. After mounting with Duolink mounting medium, samples were examined using a Zeiss 510 Meta (NLO) Confocal Microscope (Carl Zeiss).

Duolink in situ PLA was performed as previously described [23 (link)]. Duolink in situ PLA analysis was performed according to the manufacturer’s instructions (Olink Biosciences, Watertown, MA). Briefly, paraformaldehyde-fixed cells were washed with PBS, incubated for 10 min in 0.1% (volume/volume) Triton X-100, washed, and blocked with blocking solution. Primary antibodies, against ARID1A and PGR, were applied, and the cells were incubated with PLUS and MINUS secondary PLA probes against rabbit and mouse IgGs. The incubation was followed by hybridization and ligation, and then amplification was performed. After mounting with Duolink mounting medium, samples were examined using a Zeiss 510 Meta (NLO) Confocal Microscope (Carl Zeiss Microscopy, Thornwood, NY).

Ishikawa cells were plated at 20 mm glass coverslip and transfected with FLAG-tagged MIG-6 or/and V5-tagged ERK2 plasmids. After incubation for 24 h, cells were fixed for 30 min in cold 4% (weight/volume) paraformaldehyde, and permeabilized for 5 min in 1/40 Triton X-100. Primary antibodies to FLAG (Sigma Aldrich) and V5 (Bethyl Laboratories) were added and overnight at 4°C. After three washes in PBS, coverslips were incubated for 1 h at 37°C with Alexa Fluor 555 donkey anti-rabbit and Alexa Fluor 488 donkey anti-mouse (Invitrogen). Cells were then mounted with mounting media containing 4, 6-diamino-2-phenylindole (Invitrogen) for fluorescence on glass slides and visualized using a Zeiss 510 Meta (NLO) Confocal Microscope (Carl Zeiss).