Anti dig pod antibody

A DNA fragment encoding part of Papp-aa sequence was amplified using primers in Supplementary file 1. The PCR product was cloned in pGEM-T easy plasmid. The Digoxygenin-UTP labeled sense and antisense riboprobes were synthesized as previously reported (Wang et al., 2009 (link)). Zebrafish larvae were fixed in 4% paraformaldehyde, permeabilized in methanol, and analyzed by whole mount immunostaining or in situ hybridization analysis as described previously (Dai et al., 2014 (link)).
For double color in situ hybridization and immunostaining, papp-aa mRNA signal was detected using an anti-DIG-POD antibody (Roche), followed by Alexa 488 Tyramide Signal Amplification (Invitrogen). After in situ hybridization analysis, the stained larvae were washed in 1xPBST then incubated with a GFP antibody overnight at 4°C. Larvae were then stained with a Cy3 conjugated Goat anti-Rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA). Images were acquired using a Nikon Eclipse E600 Fluorescence Microscope with PMCapture Pro six software.

We dissected the ovaries and testes of 3-days old flies in PBT, which is the ideal age for optimal visualization of the different cells from ovaries. We followed the protocol described in [61 (link)]. The Helena antisense RNA probe was a 984-pb fragment corresponding to the pol-like gene (primers HelenaF1 and HelMojR1A), which included T7 and SP6 promoter sites. It was labelled by in vitro transcription of SP6/T7 using DIG RNA Labelling Kit (Roche). Labelled probes were detected using anti-DIG POD antibody (Roche) and fluorescence amplification (TSA PLUS Cyanine3 kit, PerkinElmer), visualized with a TCS-SP5 Leica confocal scanning laser microscope.

Double fluorescent in situ hybridisations were performed with a combination of DIG-labelled (neurod and crx) and fluorescein-labelled (gnat1 and gnat2) riboprobes. The protocol was similar to that described in the previous section for cryosections, with the following modifications: incubation at 4°C overnight with anti-fluorescein-POD antibody (Roche, 11426346910; 1:2000) was followed by incubation for 1 h at room temperature in TSA Plus Fluorescein Solution (PerkinElmer); inactivation of the first peroxidase was for 30 min in methanol/3% H₂O₂; incubation at 4°C overnight with anti-DIG-POD antibody (Roche, 11093274910; 1:2000) was followed by incubation for 1 h at room temperature in TSA Plus Cyanine 5 Solution (PerkinElmer). Sections were imaged on a Leica TCS SPE confocal microscope.

The FISH protocol was adapted from Jülich et al. (2005) (link) and performed as described in Turner et al. (2016) (link). Digoxigenin probes were made by standard protocols and were detected using the anti-DIG POD antibody (Roche, 1:1,000) and stained using Cy3-tyramide substrate (Perkin Elmer, 1:50 in amplification buffer). After staining, antibody labeling for GFP was performed as above using rabbit anti-green fluorescent protein (GFP, Torrey Pines Biolabs, dilution 1:1,000) and goat anti-rabbit Alexa Fluor 488 (Invitrogen, dilution 1:200) antibodies without further PK digestion. Cell nuclei were labeled with TOTO3-iodide (Invitrogen, dilution 1:5,000). Embryos were mounted in 1% agarose in 80% glycerol/PBS solution and imaged on a Leica SP8 confocal microscope.

Fluorescence in situ hybridization was performed as previously described using lef1 probes (He et al., 2020 (link)). Briefly, fish with internal organs and hands removed were fixed overnight in 4% paraformaldehyde. kidneys were harvested and embedded in OCT to obtain frozen-sections at 100 μm. Sections permeabilized with proteinase K (10 μg/mL, Roche) in PBT for 20 min with rocking. Digoxigenin-labeled riboprobes were generated from cDNA fragments comprising the sequences of zebrafish lef1 probe (F: 5′-ATGCCGCAGTTGTCAGGTG-3′, R: 5′-CGCTTTCCTCCATTGTTCAGATG-3′). Hybridization was performed as previously described (He et al., 2020 (link)). Anti-DIG POD antibody (11207733910, Roche) and TSA plus fluorescein system (NEL741001KT, PerkinElmer) were used to detect the probe. Fluorescent intensities per unit area were measured using ImageJ.

Ovaries of 3-days old flies (which is the ideal age for optimal visualization of the different cells from ovaries) were dissected in PBT, following the protocol described in [37 (link)]. Antisense RNA probes for the 9 genes (see Supplementary file 4 for probes details) of the piRNA pathway, including T7 and SP6 promoter sites, were labeled by in vitro transcription of SP6/T7 using the DIG RNA Labeling Kit (Roche) and used to detect gene expression in ovaries. Hybridization signal was detected using the anti-DIG POD antibody (Roche) and fluorescence amplification (TSA PLUS Cyanine3 kit, PerkinElmer), and visualized with an Olympus Fluoview 1000 confocal scanning laser microscope.

Intact and regenerating larvae of P. miniata at the indicated time points were fixed in a solution of 4% paraformaldehyde in MOPS-fix buffer (0.1  M MOPS pH 7.5, 2  mM MgSO₄, 1  mM EGTA, and 0.8 M NaCl) for 90  min at room temperature and transferred into 70% ethanol for long-term storage at − 20  °C. Double FISH experiments were performed as previously described (McCauley et al., 2013 (link)) using digoxigenin-labeled antisense RNA probes (final concentration of 0.1–0.2 ng/mL) and dinitrophenol-labeled antisense RNA probes (final concentration of 0.1–0.2 ng/mL). Hybridized probes were detected using anti-DIG-POD antibody (1:1000, Roche, RRID:AB_514500), anti-DNP-HRP antibody (1:1000, Perkin Elmer, RRID:AB_2629439) and tyramide signal amplification (Perkin Elmer). A 1:100 dilution of Cy3 or FITC labeled tyramide in an amplification buffer was used to treat larvae for 7 min at room temperature in the dark. A Cy3- or FITC-labeled tyramide was deposited near the hybridized probe in a horseradish peroxidase mediated reaction. This allowed for fluorescence detection of labeled probes. During PBST washes, larvae were incubated for a total of 20 min in solution with 1:10,000 dilution of 10.9 mM DAPI, followed by PBST washes. Larvae were photographed with Zeiss 880 Laser Scanning Microscope at ×200 magnification with 405 nm, 488 nm, and 560 nm channels in Z-stack settings.