Dmrxa fluorescence microscope

To analyze meiotic nuclear divisions in DAPI-stained cells, a Leica DMRXA fluorescence microscope equipped with a Hamamatsu Orca-AG CCD camera and a 63X 1.4 NA objective was used. Bright-field microscopy images of asci were captured with a Nikon Eclipse 90i microscope controlled with MetaMorph software and equipped with a Hamamatsu Orca-AG CCD camera and a PlanApo VC 63X 1.4 NA objective.

Worms were dissected and processed as described before (14 (link),31 (link)) and treated with respective primary antibody overnight and secondary antibody for 4 h at room temperature. Rabbit anti-H3K4me3 (1:1000) was purchased from Abcam (ab8580). Mouse monoclonal antibody against H3K4me2 (CMA303; 1:20) were gifts from Dr. Hiroshi Kimura (Osaka University, Japan). Mouse monoclonal antibody against GFP (1:500) was purchased from Millipore. Rabbit anti-TRA-1 (1:100) antibodies were generous gifts from Drs. David Zarkower (University of Minnesota) and Andrew Spence (University Toronto). 4',6-Diamidino-2-Phenylindole (DAPI) (Sigma, 2 μg/μl) was used to counter-stain deoxyribonucleic acid (DNA). All secondary antibodies were purchased from Molecular Probes and were used at 1:500 dilutions: goat anti-mouse IgG (Alexafluor 488); goat anti-rabbit IgG (Alexafluor 594), donkey anti-rabbit IgG (Alexafluor 488); donkey anti-mouse IgG (Alexafluor 594). Worms were mounted in anti-fade reagent (Prolong Gold, Molecular Probes). Images were collected using a Leica DMRXA fluorescence microscope and analyzed with Simple PCI software (Hamamatsu Photonics).