Dig labeled antisense rna probes

Embryos were fixed in 4% paraformaldehyde at 4°C and gradually dehydrated in methanol. Whole-mount in situ hybridizations were performed using DIG-labeled anti-sense RNA probes (made with labeling kit: Roche, 11175033910, Basel, Switzerland) to sizzled [76 (link)], foxi1 [37 (link)], and bambia. Probes were visualized with anti-DIG-Horseradish Peroxidase (Roche, 11207733910, Basel, Switzerland) developed with TSA Plus Cyanine 3 kits using a 1:50 dilution (Perkin Elmer, NEL744001KT, Waltham, Massachusetts, USA), and nuclei were stained with 1:1,000 dilution of Sytox Green. Embryos were cleared, mounted, and imaged as described for immunofluorescence. Images were analyzed using the same MATLAB algorithm, except fluorescent intensity was extracted from a 25-pixel sphere from the center point of each nucleus to include the cytoplasmic staining.

For whole-mount immunohistochemistry, planarians were stained using the following dilutions of antibodies: 1/2000 rabbit anti-planarian arrestin^{16 (link)}, 1:2000 rabbit anti-planarian GAD^{28 (link)}, 1:2000 mouse anti-planarian TPH^{31 (link)}. The plasmids pBluescript SK (−) containing planarian chat^{59 (link)}, gad^{28 (link)}, GABAA-RBa, GABAB-Ra, opsin^{6 (link)}, snap25^{24 (link)}, syt^{60 (link)}, tbh^{61 (link)}, and th^{62 (link)} cDNAs were used as templates for synthesizing digoxigenin (DIG)-labeled antisense RNA probes (Roche Diagnostics). Planarians were treated with 1% HNO₃, 50 mM MgCl₂ solution for 5 min at room temperature and fixed in 4% paraformaldehyde, 5% methanol, 50% PBS solution for 30 min at room temperture. Fixed animals were subjected to in situ hybridization with appropriate probes. For visualization of fluorescent color a TSA kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Cell nuclei were labeled with Hoechst 33342. Fluorescence was detected with a confocal laser scanning microscope (FV10i, Olympus).