For DAPI staining, cells were fixed with 70% cold ethanol, washed with PBS, and resuspended in PBS + 0.2 µg/ml DAPI (Sigma-Aldrich).
Immunofluorescence microscopy was performed as described previously (Hagan and Hyams, 1988 (link)). In brief, 10 ml of exponentially growing culture was fixed with freshly prepared 30% paraformaldehyde (294474L; VWR) in PEM buffer (0.1 M Pipes, 2 mM EGTA, and 1 mM MgSO4, pH 6.9) plus glutaraldehyde (G6257; Sigma-Aldrich) at a final concentration of 0.2%, followed by 1-h incubation at room temperature. After washing three times with PEM, the cell wall was digested during 1 h at 37ºC with 2.5 mg/ml zymolyase 20T (120491-1; AMSBIO) in PEMS (PEM + 1.2 M sorbitol); cells were permeabilized with 1% Triton X-100 and quenched with 1 mg/ml sodium borohydride. After washing twice with PEM, cell pellet was resuspended in PEMBAL buffer (PEM buffer, 0.1% sodium azide, and 1% BSA) before antibody incubation. Primary antibody was mouse anti-Myc (9E10; Santa Cruz), used at a dilution of 1:100 for mts2-8Myc and 1:200 for mts4-13Myc. The secondary antibody was goat Alexa Fluor 488–tagged anti–mouse (A11029l; Invitrogen, Molecular Probes), used at a dilution of 1:1,000.
Immunofluorescence microscopy was performed as described previously (Hagan and Hyams, 1988 (link)). In brief, 10 ml of exponentially growing culture was fixed with freshly prepared 30% paraformaldehyde (294474L; VWR) in PEM buffer (0.1 M Pipes, 2 mM EGTA, and 1 mM MgSO4, pH 6.9) plus glutaraldehyde (G6257; Sigma-Aldrich) at a final concentration of 0.2%, followed by 1-h incubation at room temperature. After washing three times with PEM, the cell wall was digested during 1 h at 37ºC with 2.5 mg/ml zymolyase 20T (120491-1; AMSBIO) in PEMS (PEM + 1.2 M sorbitol); cells were permeabilized with 1% Triton X-100 and quenched with 1 mg/ml sodium borohydride. After washing twice with PEM, cell pellet was resuspended in PEMBAL buffer (PEM buffer, 0.1% sodium azide, and 1% BSA) before antibody incubation. Primary antibody was mouse anti-Myc (9E10; Santa Cruz), used at a dilution of 1:100 for mts2-8Myc and 1:200 for mts4-13Myc. The secondary antibody was goat Alexa Fluor 488–tagged anti–mouse (A11029l; Invitrogen, Molecular Probes), used at a dilution of 1:1,000.