Study identifies two genes linked to microcephaly northwesternu NatureComms
) or 3 dpf and fixed overnight in 4% paraformaldehyde at 4 °C. Embryos were then dehydrated in methanol at −20 °C for 2 h and gradually rehydrated in methanol in PBS and 0.1% Tween in the following percent volume/volume ratios: 75/25; 50/50; 25/75 for 10 min each at RT. Embryos were bleached for 12 min in a solution of 9 ml PBST + 1 ml H+ 0.05 g KOH before proteinase K treatment and fixation in 4% PFA for 20 min at RT.
Fixed cells were then stained with primary antibodies specific to γH2AX , CENPA , 53BP1 , CENPF/Mitosin , α-Tubulin , PCNT , and RAD51 , and with secondary antibodies: anti-rabbit IgG Alexa Fluor 488 and anti-mouse IgG Alexa Fluor 594 . Cells were then stained with DAPI and visualized with aTo visualize DNA replication, cells were incubated in medium containing 10 μM EdU for 30–45 min before harvesting.
Following harvesting, cells were washed with PBS and resuspended to a concentration of 500,000 cells/ml in PBS, and then lysed in lysis buffer directly on glass microscope slides. DNA fibers were spread down the slide by gravity, fixed in methanol/acetic acid and denatured with 2.5 M HCl. The thymidine analogs, CldU and IdU, were detected via rat anti-BrdU antibody and mouse anti-BrdU antibody respectively, and secondary antibodies conjugated to Alexa Fluor 594 or Alexa Fluor 488 .
To prepare Giemsa-stained metaphase spreads from peripheral blood, whole blood was diluted in RPMI-1640 and 180 μg/ml PHA was added for 48–72 h at 37 °C. 4 h prior to harvesting 0.2 mg/ml colcemid was added. The cells were pelleted and then subjected to hypotonic shock for 10 min at 37 °C in hypotonic buffer . Finally, the cells were then fixed in methanol/acetic-acid solution and processed as described above.
For analyses of telomere sister chromatid exchange, LCLs were cultured in the presence of BrdU:BrdC and 2.5 mM BrdC ) for 10 h prior to harvesting. KaryoMAX colcemid was added at a concentration of 0.1 μg/mL during the last 2 h. Cells were collected and washed in 75 mM KCl. Cells were then fixedin methanol:acetic acid by adding fixative solution dropwise with constant gentle agitation by vortex.
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