Dig ap rna labeling kit

A PCR product was generated using gsdf or inha gene-specific primers (Table 3) for the synthesis of cRNA-probes for ISH. The PCR products were sequenced following a PCR cleanup using illustra ExoProStar 1-Step (GE Healthcare Life Sciences), according to the manufacturer’s instructions. The returned sequences were blasted in the NCBI database (https://www.ncbi.nlm.nih.gov/) against several species, confirming that the primers had amplified the gsdf and inha genes. ISH cRNA antisense and sense probes were synthesized from 1 μg PCR product applying primers containing Sp6 or T7 sequence, respectively (Table 3), together with a digoxigenin-alkaline phosphatase (DIG-AP) RNA Labeling Kit (SP6/T7) (Roche Diagnostics). The probes were precipitated, washed, and resuspended in MilliQ water as described by Weltzien et al. [73 (link)]. Probe size and quality were checked with a Bioanalyzer (Agilent Technologies), and the DIG incorporation in the probes was inspected by performing a spot-test. ISH by DIG-AP was performed as described by Weltzien et al. [73 (link)].

PCR was done with vgll3-specific primers with added Sp6/T7 sequences, and cRNA probes for in situ hybridization were produced from gDNA with DIG-AP RNA Labeling Kit (Sp6/T7, Roche Diagnostics). Primers used for generating the probe for vgll3 are listed in Supplementary Table S2. Probes were precipitated, washed and resuspended as previously described^{37 (link)}. Antisense and sense were produced by T7 and Sp6 polymerases, respectively. Quality of the probes was inspected with BioAnalyzer (Agilent), and DIG incorporation estimated with a spot-test. In situ hybridization with digoxigenin-alkaline phosphatase (DIG-AP), including preparation of tissue, was done on cryosections as previously described^{37 (link)} on prepubertal testis and ovary, and from germ cell-free testis (described elsewhere^{25 (link),38 (link)}).

Six larvae from each replicate were pooled and fixed in 4% PBS buffered paraformaldehyde for 24 hrs at 4.0 °C, processed using Histokinette 2000 (Reichert-Jung), and embedded in paraffin wax within three days to preserve the RNA and tissue morphology. Sample preparation was always performed under RNase free conditions. Serial sectioning (3 μm) of larvae was performed for morphological analysis or in situ hybridization using a Leica RM 225 microtome (Leica Microsystems).
For in situ hybridization (ISH), the Atlantic haddock cyp1a cDNA was amplified using PCR containing Sp6 and T7 primers in the forward and reverse primers, respectively (Cyp1a_FW_Sp6: 5′-ATTTAGGTGACACTATAGCATCTTCCAGATCCAGATCG-3′ Cyp1a_RV_T7: 5′-TAATACGACTCACTATAGGGCATGAACCTCTTCATGGTGG-3′). The PCR product of 501 bp was used as a template for synthesizing the sense and antisense cRNA probes by the Sp6 and T7 RNA polymerase, respectively. The digoxigenin (DIG) labeling was performed using the DIG-AP RNA labeling kit (Roche Molecular Biochemicals) following the manufacturers protocol.
The in situ hybridization was carried out as described by Weltzien et al.82 (link) with some modifications83 . Hybridization was always carried out with sense and antisense probes on adjacent sections, and under RNase free conditions.