In situ Hybridization of Nematode Ab-cb-1

In situ hybridization was performed as described previously [1 (link),18 (link)]. Approximately 10,000 nematodes of mixed stages of the Ab-S24 population were collected and concentrated into 30–50 μl. The nematodes were fixed in 3% paraformaldehyde for 18 h at 5°C and then at 22°C for 4 h. DIG-labeled sense and antisense RNA probes (Roche, Germany) were synthesized using sense primers (CB-IN-T7S1, CB-IN-A1) and antisense primers (RB-IN-TA1, CB-IN-S1) (Table 1) based on the full-length cDNA of Ab-cb-1. After adding the obtained DIG-labeled RNA probes, the hybridization solution containing nematodes was rotated for 12 h at 47°C. The results were examined and photographed by differential interference microscopy.