Mouse monoclonal anti α tubulin antibody dm1a

10–15 worms were dissected in 7 µl of blastomere culture solution on subbing solution–coated slides using a 21-gauge needle to release embryos. An 18 × 18-mm coverslip was placed onto the drop, and the slide was frozen on a block of metal precooled on packed dry ice. After 20 min, the coverslip was flicked off, and the slide was plunged into −20°C methanol for 20 min. Slides were rehydrated in PBS for 5 min and incubated overnight at 4°C with 30 µl of primary antibodies in 3% BSA in PBS in a wet chamber. Affinity-purified rabbit anti-MEI-1 antibody was used at a dilution of 1:500, mouse monoclonal anti-α-tubulin antibody (DM1A; Sigma-Aldrich) was used at 1:400, and the secondary antibodies were coupled to the fluorophores Alexa Fluor 488 or 543 (Molecular Probes) used at 1:1,000. Embryos were mounted in Vectashield mounting medium with DAPI. Fixed embryos were imaged using a spinning disk confocal X1 microscope with 63× objective. Captured images were processed using ImageJ and Photoshop.