T7 or sp6 rna polymerase

In-situ hybridization was basically performed as previously described (Ibarra-Soria et al., 2017 (link)). Adult 12-week-old male C57BL/6J mice anesthetized, and then perfused with 4% paraformaldehyde. The snouts containing the OM were dissected out, decalcified in RNase-free 0.45M EDTA solution (in 1× PBS) for two weeks – the bone and tissue encapsulating the OM are necessary to preserve the OM tissue integrity during the ISH. Next, the decalcified snouts were cryoprotected in RNase-free 30% sucrose solution (1× PBS), dried, embedded in OCT embedding medium, and frozen at −80°C. Sequential 16 μm sections were prepared with a cryostat and the sections were hybridized to digoxigenin-labeled cRNA probes prepared from the different genes using the following oligonucleotides: Cytl (5′-AAAGACACTACCTCTGTTGCTGCTG-3′ and 5′-GTAAGCAGAGACCAGAAAGAAGAGTG-3′), Moxd2 (5′-TGTACCTTTCTCCCACTCCCTATTGTC-3′ and 5′-CCCATGCAACTGGAGATGTTAATTCTG-3′), Olfr309 (5′-TACAATGGCCTATGACCGCTATGTG-3′ and 5′-TCCTGACTGCATCTCTTTGTTCCTG-3′), Olfr727 (5′-CGCTATGTTGCAATATGCAAGCCTC-3′ and 5′-GCTTTGACATTGCTGCTTTCACCTC-3′), and Olfr618 (5′-CATGAACCAATGTACCTTTTCCTCTC-3′ and 5′-AAACCTGTCTTGAATTTGCTTTGTC-3′). The PCR products were cloned into pGEM-T Easy vector and the probes were obtained by in vitro transcription of the plasmids, using SP6 or T7 RNA Polymerases (Roche) and DIG RNA Labeling mix (Roche).

We designed antisense probes to bind to different variants of the OT mRNA. Antisense probes for the OT mRNA were synthesized from the BF OT cDNA (Figure S1 and Table S2). Corresponding sense probes were also synthesized for control ISH (Figure S2). BF OT cDNA fragments were inserted into the pGEM‐T easy vectors (Promega). The plasmids were digested with restriction enzymes (NcoI or SpeI) to release the fragment; probes were synthesized using SP6 or T7 RNA polymerase (Roche Diagnostics) with digoxigenin‐labeling mix (Roche Diagnostics).
Frozen brains were cut in 20‐μm coronal sections on a cryostat (Leica Microsystems, Wetzlar, Germany). Every 13th section through the hypothalamus from each animal was mounted on 3‐aminopropyltriethoxysilane‐coated slides and stored at −80°C until use. ISH was performed as described previously,⁴¹ except that sections were postfixed in 4% paraformaldehyde for 10 min, proteinase K treatment was omitted and color development of alkaline phosphatase activity was carried out for 1 h with nitro blue tetrazolium chloride (Roche Diagnostics) and 5‐bromo‐4‐chloro‐3‐indolyl phosphate (Roche Diagnostics) in detection buffer. Images of sections were captured with a LeicaMC170 HD camera attached to a Leica DM500 microscope (Leica Microsystems).