For analysis of cytoophidium, the cells grown in 13-mm round coverslips were fixed with 4% paraformaldehyde and probed with antibodies in an indirect immunofluorescence assay, as previously described (Keppeke et al., 2018 (link); Keppeke et al., 2022 (link)).
Primary antibodies used: rabbit polyclonal anti-IMPDH2 antibody (12948-1-AP, ProteinTech), mouse anti-Myc monoclonal antibody 9E10 (sc-40, Santa Cruz Biotech), and mouse anti-Flag monoclonal antibody clone M2 (F1804, Sigma). Secondary antibodies used: Cy3-conjugated donkey anti-mouse IgG (#715-165-151, Jackson ImmunoResearch) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (#A-21206, Invitrogen). After immunofluorescence labeling, the cells were covered with VECTASHIELD containing DAPI (Vector Labs, USA), and the images were captured either by a fluorescence microscope with 200 × or 400 × magnification (Axio Imager. M2, Carl Zeiss, Germany) or by a confocal microscope (LSM 800, Carl Zeiss, Germany).
Primary antibodies used: rabbit polyclonal anti-IMPDH2 antibody (12948-1-AP, ProteinTech), mouse anti-Myc monoclonal antibody 9E10 (sc-40, Santa Cruz Biotech), and mouse anti-Flag monoclonal antibody clone M2 (F1804, Sigma). Secondary antibodies used: Cy3-conjugated donkey anti-mouse IgG (#715-165-151, Jackson ImmunoResearch) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (#A-21206, Invitrogen). After immunofluorescence labeling, the cells were covered with VECTASHIELD containing DAPI (Vector Labs, USA), and the images were captured either by a fluorescence microscope with 200 × or 400 × magnification (Axio Imager. M2, Carl Zeiss, Germany) or by a confocal microscope (LSM 800, Carl Zeiss, Germany).
Free full text: Click here
