Dig labeled sense and antisense rna probes

Gene-specific primers (miFARp_F and miFARp_R) were used for localization of expression of Mi-far-1 mRNA by performing in situ hybridization as described in Kimber et al. (2002) (link). Around 20,000 J2s were concentrated to a 10–15 μl pellet and fixed in 2% paraformaldehyde at 4°C for 18 h, followed by 4 h incubation at room temperature and cut into sections. The gene specific primers miFARp_F and miFARp_R were used to amplify DIG-labeled sense and antisense RNA probes (Roche, Germany) from cDNA of M. incognita J2s. DIG-labeled sense or antisense RNA probe was added to the hybridization solution containing the nematode sections, and then rotated at 50°C for 12 h. After hybridization, the nematodes were stained and photographs were taken with Zeiss Axiocam compound microscope.

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.