Dig labelling mix

For each IgLON member, digoxigenin-labeled cRNA probes with either antisense or sense orientation were prepared by in vitro transcription using DIG labelling mix (Sigma,USA). Probe sequences were PCR-amplified from cDNAs prepared from mouse brain cDNA using primers designed based on sequences from the Allen Brain Atlas and Gene Paint Database (see Supplemental Material for probe sequences) and cloned into a pBluescript vector. cRNA probes were designed to detect expression of both the 1a and 1b transcripts for IgLON1, IgLON2, and IgLON3. Following in vitro transcription, the riboprobes were partially hydrolyzed in a 10 mM DTT and 200 mM NaHCO₃/Na2CO₃ solution (pH 11) for 25 min at 60 °C^{79 (link)}. Hydrolysis was stopped by addition of 100 mM acetic acid, and cRNA probes were precipitated by addition of 1/10th volume 4 M LiCl and ethanol. Precipitated cRNA fragments were resuspended in diethylpyrocarbonate (DEPC)-treated water and stored at − 80 °C.

Hydra polyps were treated with target inhibitors for 8 h and decapitated. After decapitation, the inhibitor treatment continued until the target time points of 1, 2, 4, and 8 hpa. The polyps were then relaxed by treatment with 2% urethane/HM for 2 min and fixed using 4% PFA/HM at 4°C overnight. Digoxigenin-labelled RNA probes for Hv_Bra1 were prepared by in vitro transcriptions from templates amplified from a recombinant pCR Blunt II TOPO (Cat # 450031; Invitrogen) plasmid containing the Hv_Bra1 gene using PCR. (DIG Labelling Mix, Cat # 1277073910; Sigma-Aldrich; SP6 RNA Polymerase, Cat # 10810274001; Sigma-Aldrich; T7 RNA Polymerase, Cat # 10881767001; Sigma-Aldrich). Whole-mount in situ hybridization was performed on the polyps as described previously (Martinez et al, 1997 (link)) with the following changes. Treatment with proteinase-K was performed for 5 min, and heat inactivation of the endogenous alkaline phosphatases was done at 70°C for 15 min in 1X SSC. Digoxigenin-labelled RNA probe at a concentration of 150 ng/ml was used for hybridization at 59°C. The post-hybridization washes were performed using 1X SSC-HS gradients. After staining with 50% NTMT/50% BM-purple AP substrate for 1 h at room temperature, the animals were mounted in 80% glycerol for imaging.

pBlueScript SK ( + ) vector (Addgene #212205) containing the target DNA sequence for murine Angptl2—subcloned using EcoRI/HindIII restriction sites between the T3 and T7 promoters—was linearized with the restriction enzyme MluI (R3198S, New England Biolabs) and purified through silica membrane spin columns (20021, Qiagen). The synthesis of a 980 bp anti-sense probe was then realized with 500 ng of linearized DNA incubated with DIG labelling mix (11277073910, Sigma) and T3 polymerase (M0378S, New England Biolabs). The transcription product was further treated with DNase (74204, Qiagen) to clear the probe from all residual DNA. The RNA probe was then purified through a G-50 column (CA95017-623L, Amersham) and stored at −30 °C.