Before immunolabeling, coverslips were washed twice with 1xPBS keep in one line for 5 min at room temperature (RT) and incubated with blocking solution (5% BSA, 0.03% Triton X-100, 1×PBS) for 1.5 h at RT. Peptide Topo IIα (rb12 and gp13) (Kubalová et al. 2021b (link)) and rabbit anti-grassCENH3 (Nagaki et al. 2004 (link); Houben et al. 2007 (link)) antibodies were diluted 1:100 and 1:10,000, respectively, in antibody solution (1% BSA, 0.01% Triton X-100, 1 × PBS), and incubated overnight at 4 °C. Grass-CENH3 antibodies detect both α and βCENH3 of barley (Ishii et al. 2015 (link)).
Next, coverslips were washed with 1×PBS (three times, 5 min each) at RT and incubated with secondary donkey anti-rabbit Alexa488 (1:200, #711-545-152 Jackson ImmunoResearch) and goat anti-guinea pig Alexa488 (1:200, # A11073 Invitrogen) antibodies for 1 h at 37 °C. For colocalization with Topo IIα, CENH3 was labeled with Cy3-conjugated anti-rabbit IgG (Dianova). Subsequently, coverslips were washed in 1×PBS (three times, 5 min each) at RT and immediately dehydrated in an ethanol series (70%, 85%, and 100%), each step 2 min. Afterward, the coverslips were air-dried and subjected to microscopy.
Next, coverslips were washed with 1×PBS (three times, 5 min each) at RT and incubated with secondary donkey anti-rabbit Alexa488 (1:200, #711-545-152 Jackson ImmunoResearch) and goat anti-guinea pig Alexa488 (1:200, # A11073 Invitrogen) antibodies for 1 h at 37 °C. For colocalization with Topo IIα, CENH3 was labeled with Cy3-conjugated anti-rabbit IgG (Dianova). Subsequently, coverslips were washed in 1×PBS (three times, 5 min each) at RT and immediately dehydrated in an ethanol series (70%, 85%, and 100%), each step 2 min. Afterward, the coverslips were air-dried and subjected to microscopy.
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