Sheep anti dig antibody conjugated to alkaline phosphatase

For 5-HT1A receptor in situ hybridization, the head of the animals was fixed by immersion in 4% PFA in 0.05 M Tris-buffered saline (TBS; pH 7.4) for 12 h. Then, the brains were dissected out, washed and embedded in Neg 50™ (Microm International GmbH, Walldorf, Germany), frozen in liquid-nitrogen-cooled isopentane, sectioned on a cryostat in the horizontal plane (14 μm thick) and mounted on Superfrost Plus glass slides (Menzel, Braunschweig, Germany). In situ hybridization with a specific riboprobe for the 5-HT1A receptor (GenBank accession number KU314442.1) was conducted as previously described (Cornide-Petronio et al., 2013 (link), 2014 (link)). Briefly, brain sections were incubated with the sea lamprey 5-HT1A receptor DIG-labelled probe at 70°C and treated with RNAse A (Invitrogen, Massachusetts, USA) in the post-hybridization washes. Then, the sections were incubated with a sheep anti-DIG antibody conjugated to alkaline phosphatase (1:2000; Roche, Mannhein, Germany) overnight. Staining was conducted in BM Purple (Roche) at 37°C. In situ hybridization experiments were performed in parallel with animals from the different experimental groups (control, 1 wpl and 4 wpl) and the colorimetric reaction was stopped simultaneously for all sections from the different groups of animals.

For gabab1 in situ hybridization, the head of the animals was fixed by immersion in 4% paraformaldehyde (PFA) in 0.05 M Tris-buffered saline (TBS; pH 7.4) for 12 h. Then, the brains were dissected out, washed and embedded in Neg 50^TM (Microm International GmbH, Walldorf, Germany), frozen in liquid nitrogen-cooled isopentane, sectioned on a cryostat in the transverse plane (14 μm thick) and mounted on Superfrost Plus glass slides (Menzel, Braunschweig, Germany). In situ hybridization with a specific riboprobe for the gabab1 subunit of the sea lamprey gabab receptor (GenBank accession number KX655780; see Suppl. Figure 1) was conducted as previously described^{42 (link)}. Briefly, brain sections were incubated with the sea lamprey gabab1 DIG-labelled probe at 70 °C and treated with RNAse A (Invitrogen, Massachusetts, USA) in the post-hybridization washes. Then, the sections were incubated with a sheep anti-DIG antibody conjugated to alkaline phosphatase (1:2000; Roche, Mannhein, Germany) overnight. Staining was conducted in BM Purple (Roche) at 37 °C. In situ hybridization experiments were performed in parallel with animals from the different experimental groups (control, 1 wpl, 2 wpl and 4 wpl) and the colorimetric reaction was stopped simultaneously for all sections from the different groups of animals.