Hnpp fluorescent detection set

Antennae of male moths were embedded with NEG 50^TM compound (Thermo Fisher, Cheshire, UK) in Tissue-Tek Cryomold (CITOTEST, HaiMen, China) and frozen at −60 °C rapidly. Longitudinal sections (12 μm) of antennae were sectioned with cryostat microtome (LEICA CM1850) at −23 °C and pasted on Superfrost Plus slides (Thermo Fisher, New Hampshire, U.S.A.), then air-dried at room temperature for about 20 min.
DIG-labeled or Biotin-labeled antisense probes were synthesized with T7/SP6 DIG/Biotin RNA Labeling system (ROCHE, Mannheim, Germany). Two-colour fluorescence in situ hybridization was performed as described in references45 (link)46 (link) DIG-labeled RNA probes were detected by anti-DIG alkaline phosphatase-conjugated antibody (ROCHE) and visualized by the using HNPP Fluorescent Detection Set (ROCHE). For biotin-labeled RNA probes, the tyramide-signal amplification (TSA) kit (PerkinElmer, Boston, U.S.A.) was used. Sections were mounted using SlowFade Gold Antifade reagent (Molecular Probes, CA, U.S.A.). The slides were sealed with nail polish. Finally, the labeled signals were imaged and analyzed by Zeiss LSM780 laser scanning confocal microscope. Images were adjusted and arranged with Photoshop CS6 (Adobe) and Illustrator CS5 (Adobe). The contents of images were not modified except for the brightness or contrast.

Preparation of the RNA probes and in situ hybridization were carried out as previously described [8 (link)]. Digoxigenin (DIG)- and fluorescein-labeled antisense and sense RNA probes for lamprey parapinopsin and parietopsin mRNAs were synthesized using the DIG RNA-labeling kit and fluorescein RNA-labeling kit (Roche), respectively. Sections were pre-treated with proteinase K and hybridized with each RNA probe in ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion). The pineal sections hybridized with DIG-labeled probes were incubated with horseradish peroxidase (HRP)-conjugated anti-DIG antibody (Roche) and subsequently treated with the TSA plus DNP (HRP) system (Perkin Elmer), followed by incubation with Alexa 488-conjugated anti-DNP antibody. The fluorescein-labeled probes were detected through incubation with alkaline phosphatase-conjugated anti-fluorescein antibody (Roche) followed by a color reaction using the HNPP Fluorescent Detection Set (Roche).

Cells were fixed with 4% (w/v) paraformaldehyde in PBS for 10 min, permeabilized with 0.1% (v/v) Triton X-100 in PBS for 15 min, and blocked in 1% normal donkey serum for 1 h. Cells were incubated with primary antibodies for 1 h, washed with PBS, and incubated with appropriate Alexa Fluor-conjugated secondary antibodies for 1 h. Nuclei were counterstained with 20 µM Hoechst 33342 (Dojindo, H342) for 10 min. Primary antibodies were as follows: Nanog (1∶800; Cell Signaling, 4903), Oct4 (1∶100; SantaCruz, sc-5279), Sox2 (1∶100; R&D Systems, MAB2018), SSEA-4 (1∶100; Stemgent, 09-0006), Tra-1-60 (1∶100; Stemgent, 09-0010), Tra-1-81 (1∶100; Stemgent, 09-0011), Pax6 (1∶200; BD Biosciences, 561462), Sox1 (1∶100; Stemgent, 09-0084), T/Brachyury (1∶200; SantaCruz, sc-17745), Tuj1 (1∶500; Millipore, MAB1637), MAP2 (1∶500; Millipore, AB5622), DCX (1∶500; Abcam, ab18723), and CTIP2 (1∶500; Abcam, ab18465). Alkaline phosphatase staining was performed using the HNPP Fluorescent Detection Set (Roche).

After hybridization, sections were washed twice in 0.2× SSC at 63°C for 30 min, blocked with the Streptavidin/Biotin Blocking Kit (Vector Laboratories), incubated with POD-conjugated anti-FLU antibodies (Roche, #11426346910; 1:300) and alkaline phosphatase–conjugated anti-DIG antibodies (Roche, #11093274910; 1:300) at RT for 2 hours, and then treated using the TSA Plus Biotin Kit (PerkinElmer). Sections were next incubated with DAPI (0.5 μg ml⁻¹) and Alexa Fluor 488–streptavidin (Thermo Fisher Scientific; 1:1000) at RT for 30 min, incubated with HNPP Fluorescent Detection Set (Roche) at RT for 1 hour, and then coverslipped with Fluoromount-G.