Cloning and in situ Hybridization of fhl2a and fhl2b in Astatotilapia burtoni

A. burtoni fhl2a and fhl2b coding fragments were amplified by PCR (for primer information, see Supplementary Table 3) using Phusion Master Mix with High Fidelity buffer (New England BioLabs, USA) following the manufacturer’s guidelines. These fragments were cloned into pCR4-TOPO TA vector using the TOPO TA cloning kit (Invitrogen). Plasmid extractions were done with GenElute Plasmid Miniprep Kit (Sigma-Aldrich). RNA probes were synthetized with the DIG RNA labelling kit (SP6/T7) (Roche). The insertion and direction of the fragments was confirmed by Sanger sequencing using M13 primers (available with the cloning kit) and BigDye terminator reaction chemistry (Applied Biosystems) on an AB3130xl Genetic Analyzer (Applied Biosystems). In situ hybridization was performed in 12 fins from A. burtoni males, six for fhl2a and six for fhl2b. The protocol was executed as described in ref. 16 (link), except for an intermediate proteinase K treatment (20 min at a final concentration of 15 μg ml⁻¹) and for the hybridization temperature (65 °C).