Anti dig pod fab fragments

Paraffin tissue sections were deparaffinized and treated with 10 μg/ml proteinase K (Roche) at 37 °C for 10 min, as reported18 (link). After washing with PBS, slides were incubated with the hybridization buffer (50% formamide, 100 μg/ml salmon sperm DNA, 200 μg/ml yeast tRNA, 600 mM NaCL, 1 × Denhardt’s solution, 0.25% SDS, 1 mM EDTA) at 42 °C for 1 h. Slides were then hybridized with 20 nM DIG-labelled miR-27b probe (Exiqon) diluted in the hybridization buffer at 42 °C overnight. Slides were incubated with anti-DIG-POD Fab fragments (Roche) at 4 °C overnight and miR-27b was visualized in a staining reaction with Renaissance Tyramide Signal Amplification Fluorescence Systems (PerkinElmer). For all experiments, a negative control (i.e. staining without miR-27b probe) was included18 (link).

The sequence-specific DNA fragments of lnc196, lnc304, and lnc172 were amplified with a pair of primers whose reverse primer contained the core sequence of T7 promoter at the 5′ end (primer and lncRNA sequence information in Table S1). Digoxin-labelled antisense RNA probes were synthesized using the DIG-RNA labeling kit (Roche, Mannheim, Germany). The gonads were fixed in 4% PFA at room temperature for 2 h, washed in PBS for 3 × 10 min, and embedded with frozen section compound (Sakura Finetek, Torrance, USA). The samples were sectioned with a thickness of 10 μm. The slides were dried at 37 °C for 30 min, rehydrated, and washed in PBS three times and hybridized with DIG-labeled anti-sense probes at 60 °C for 16 h. After a series of washing steps, the slides were blocked in 2% blocking reagent (Roche, Mannheim, Germany) in MAB buffer (0.1 M Maleic acid and 0.15 M NaCl, pH 7.4) for over 2 h at room temperature and incubated in anti-DIG-POD FAB fragments (Roche, 1:2000 diluted in 1% blocking solution) and anti-vasa antibodies (abcam, ab209710, 1:400) at 4 °C overnight. After washing in PBT and PBS buffer three times, the slides were incubated in TSA™ Plus Fluorescence Systems (Red) (PerkinElmer), FITC-conjugated goat anti-rabbit IgG (H + L), and DAPI (1 μg/mL) for 1 h. The images were acquired with a Leica Stellaris 5 confocal microscope (Leica, Mannheim, Germany).

Candidate genes (Supplementary Table 6) and gene markers used to characterise the drug phenotypes were amplified by two rounds of nested PCR using gene-specific primers and a T7 universal primer on cDNA from mixed developmental stages as initial template. Riboprobes were synthesized with the T7 enzyme (Ambion’s MEGAscript kit, #AM1334) and stored in hybridisation buffer at a concentration of 50 ng/μl at −20 °C. Colorimetric and fluorescent whole mount in situ hybridisation were performed following an established protocol^{3 (link),30 (link)}. After probe washes, samples for fluorescent in situ hybridisation were incubated with anti-DIG-POD Fab fragments (ROCHE, #11633716001) and a mouse anti-acetylated α-tubulin antibody (clone 6-11B-1, Millipore-446 Sigma, #MABT868, 1:500) to co-stain cell boundaries. After signal development with a Tyramide Signal Amplification kit (Akoya Biosciences, #NEL742001KT), these samples were washed several times in PTx and treated for secondary antibody incubation as described above. To characterise in silico gene expression dynamics during development, we used an available stage-specific RNA-seq dataset³³ , representing the average expression of the two replicates with the package pheatmap v.1.0.12 available in R, were colour intensity shows the z-score value for each gene.

The OE of blunt snout bream was fixed with 4% paraformaldehyde for 24 h, dehydrated by a series of gradient ethanol baths, embedded in paraffin, and then sectioned (5 µm). Fragments (length was between 150 and 250 bp) of four genes (beta1, beta12, epsilon9, and eta28) were cloned using the primers shown in Supplementary Table S5. The PCR products were purified, and then the in situ hybridization probe was synthesized in accordance with the instructions of the manufacturer, Sigma-Aldrich, using the DIG RNA Labeling Kit (SP6/T7) (Roche Diagnostics GmbH, Mannheim, Germany, SKU: 11175025910). The steps of fluorescence in situ hybridization were performed according to the method described in Alamri et al. [70 (link)]. Anti-DIG-POD, Fab fragments (Roche Diagnostics GmbH, SKU: 11207733910) from Sigma-Aldrich were used as antibodies.