Alkaline phosphatase conjugated anti dig antibody

Embryos were fixed in 4% paraformaldehyde for 4 h, dehydrated in methanol, and stored in 100% MeOH at −20°C until use. Samples were rehydrated, pretreated with proteinase K, and hybridized with DIG-labeled RNA probes followed by washing with 2 × SSC/50% formamide three times at 70°C. The signal was detected using an alkaline phosphatase-conjugated anti-DIG antibody (11093274910; Roche). Tissues were incubated in the BM Purple alkaline phosphatase substrate (11442074001; Roche) at 4°C for several hours until the signal developed to the desired extent. Probes for each gene were generated using DIG RNA Labeling Kit (11 175 025 910; Roche). Primers for probe generation were listed in Supplementary Table 4.

Control and Tpp1 mutant 2-month brains were fixed in 4% paraformaldehyde and processed for non-radioactive in situ hybridization as described previously [54 (link), 55 (link)]. To prepare the digoxigenin (DIG)-labeled RNA probes used for ISH, cDNA fragments from Lipopolysaccharide binding protein (Lbp;) and Klotho (Kl) genes were generated by PCR and inserted into pCRII dual-promoter vector plasmids (Invitrogen) Lbp probe: nt 360–1229 of NM 008489.2 and Kl probe: nt 3505–4495 of NM 013823.2. Sequencing of the cloned gene fragments was performed with an ABI PRISM 377XL sequencer (Perkin Elmer) by the University of Chicago Cancer Center DNA sequencing facility. Riboprobes incorporating DIG-labeled nucleotides were synthesized from linearized PCR templates with SP6 or T7 RNA polymerase (Roche). Probed mRNAs were detected after hybridization with an alkaline-phosphatase-conjugated anti-DIG antibody (Roche). Alkaline phosphatase activity was detected (blue color) using BCIP and NBT substrates (Roche). Images were obtained using an Axiovert 200 microscope (Zeiss).

Single-label in situ hybridization was performed as described previously^{48 (link)}. Briefly, whole brains were fixed in 4% PFA and embedded in paraffin. Serial coronal sections of 10 μm thickness were cut and hybridized with the above-mentioned ptger4b or npba probe, which was labeled with DIG using DIG RNA Labeling Mix and T7 RNA polymerase (Roche Diagnostics). Hybridization signals were visualized using an alkaline phosphatase-conjugated anti-DIG antibody (RRID: AB_514497; Roche Diagnostics) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate (Roche Diagnostics). Color development was allowed to proceed overnight (for analysis of ptger4b expression) or was stopped within 15 min to avoid saturation (for analysis of npba expression). Brain nuclei were identified using the medaka brain atlas⁵⁰ . For quantification of npba expression in FeSP neurons, images were acquired with a virtual slide microscope (VS120; Olympus, Tokyo, Japan) and the total area of npba expression signal in the PMm/PMg was calculated using Olyvia software (Olympus).

For the construction of riboprobes for in situ hybridization analysis, we prepared two sets of each antisense or sense sequence. The probes were amplified via PCR using the primer sets listed in S4 Table and then subcloned into the pCR^®4Blunt-TOPO^® plasmid vector. Antisense or sense riboprobes were generated using these plasmids as templates in T3 or T7 RNA polymerase-directed in vitro transcription mixtures containing a digoxigenin (DIG)-labeled mix (Roche). Hind limbs were fixed in 4% PFA/PBS at 4°C overnight, then maintained in 0.5 M sucrose/PBS at 4°C for cytoprotection and subsequently embedded in O.C.T. compound. Sections with a size of 20 μm treated with 0.5 μg/ml proteinase K (Wako)/0.1% Tween 20 in PBS at 37°C for 10 min and then post-fixed in 4% PFA/PBS for 10 min and rinsed in PBS. Hybridization with DIG-labeled riboprobes was conducted overnight at 55°C in 50% formamide, 5× Denhardt’s solution, 5× SSC, and 50 μg/ml yeast tRNA and then washed with 5× SSC and 2× SSC at 55°C for 15 min each. After washing, the slides were digested with RNase A (20 μg/ml; Sigma-Aldrich) in 0.5 M NaCl/10 mM Tris-HCl pH 7.5/0.1% Tween 20 for 30 min at 37°C. Next, the slides were washed twice with 2× SSC at 42°C for 20 min. After incubation with an alkaline phosphatase-conjugated anti-DIG antibody (Roche), DIG-labeled RNA duplexes were detected with BM purple (Roche).

For Figs 3B, 5 and 7A, in situ hybridisation for D1, D2, and c-fos mRNA was performed as previously described with modification^{42 (link)}. Briefly, coronal sections were fixed in 4% PFA, digested with proteinase K (10 μg/mL) for 30 min, and post-fixed in 4% PFA. Following pre-hybridisation, sections were incubated overnight at 60 °C with DIG-labelled RNA probes. After stringent washing, blocking was performed using 10% normal sheep serum, 1% bovine serum albumin, and 0.1% Triton X-100 in PBS. Then sections were incubated overnight at 4 °C with alkaline phosphatase-conjugated anti-DIG antibody (1:1000 dilution; Roche). Sections were washed in MABT (100 mM maleic acid, 150 mM NaCl, and 0.1% Tween 20) followed by alkaline phosphatase buffer (100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 50 mM MgCl₂, 0.1% Tween 20, and 5 mM levamisole). Sections were treated with NBT/BCIP (Roche) mixture at room temperature in the dark for colour development. In Figs 5 and 7A, sections were counterstained with nuclear fast red (Vector). Then the sections were dehydrated in ethanol, cleared in Xylene, and mounted in Mount Quick (Daido Sangyo).