To generate DIG-labelled sense and antisense RNA probes of Ds_β2t, we prepared DNA templates for in vitro transcription by PCR amplification of the 5’RACE-fragment including the Sp6 or T7 promoters from pCRII_Ds β2t_5’UTR (HMMA24). Primer pairs HM#33/128and HM#41/127 were used respectively with the following PCR conditions: initial denaturation at 98 °C 3 min, followed by 35 cycles of 98 °C 30 s, 72 °C 50 s with a final elongation step of 7 min. RNA probes were synthesized using DIG-labelling kit (Thermo Fisher Scientific) according to manufacturer instructions using 200 ng of DNA as template in a total reaction mix of 10 μl. The reaction was allowed to proceed for 2 h at 37 °C followed by Turbo DNaseI treatment (Thermo Fisher Scientific) for 15 min to remove template DNA. Two microliter of 0.2 M EDTA was used to inactivate the reaction. Sense and antisense probes were precipitate and resuspended in 100 μl RNA resuspension buffer (5:3:2 H2O: 20X SSC: formaldehyde) and stored at − 80 °C.
Testes of 3–5 days old males were dissected in ice cold 1X Phosphate buffered saline (PBS) and fixed in PBF-tween (4% formaldehyde and 0.1% tween 20 in 1X PBS) for 20 min at room temperature. In situ hybridization was performed according to an established protocol [56 ] with inclusion of dehydration steps according to Zimmerman et al. [57 (link)].
Testes of 3–5 days old males were dissected in ice cold 1X Phosphate buffered saline (PBS) and fixed in PBF-tween (4% formaldehyde and 0.1% tween 20 in 1X PBS) for 20 min at room temperature. In situ hybridization was performed according to an established protocol [56 ] with inclusion of dehydration steps according to Zimmerman et al. [57 (link)].