Anti digoxigenin ap fab fragment

The whole-mount in situ hybridization (WISH) experiment was manipulated as previously recited (He et al. 2014 (link); Thisse and Thisse 2014 (link)). Briefly, after fixation and gradient dehydration, the embryos were warehoused in pure methanol at − 20 °C. Embryos were rehydrated and then digested in 20 μg/ml proteinase K for preparation of hybridization, then embryos were incubated with the probes at 65 °C overnight. After labeling, larvae were rinsed four times in a graded SSC-PBST series and then put in the blocking reagent at room temperature for 1 h. After bonding to the anti-digoxigenin-AP Fab fragment (Roche) overnight at 4 °C, the embryos were washed sufficiently and then incubated with BM purple AP substrate (Roche) for visualized staining in a dark place. The color reaction was discontinued by NTMT and washed thoroughly in PBST. For whole-mount neuromast imaging, specimens were mounted in 100% glycerol, with a Nikon fluorescence stereomicroscope. All image processing was performed with Photoshop and Illustrator software (2018, Adobe).

In situ hybridization was performed as described previously (Kraus et al., 2016 (link)) with a single change: the embryos were fixed for 1 h at room temperature in 4%PFA/PBS, washed several times in PTw (1× PBS and 0.1% Tween 20), then in 100% methanol and finally stored in 100% methanol at −20°C. Digoxigenin-labelled RNA probes were detected with anti-digoxigenin-AP Fab fragments (Roche) diluted 1:4000 in 0.5% blocking reagent (Roche) in 1× MAB. After unbound antibody was removed by a series of ten PTw washes of 10 min each, the embryos were stained with a mixture of NBT/BCIP, embedded in 86% glycerol and imaged using a Nikon 80i compound microscope equipped with the Nikon DS-Fi1 camera. For phalloidin staining, the embryos were fixed in 4%PFA/PTwTx (1× PBS, 0.1% Tween 20 and 0.2% Triton X-100) for 1 h at room temperature, washed five times with PTwTx, incubated in 100% acetone pre-cooled to −20°C for 7 min on ice and washed three more times with PTwTx. 2 µl of phalloidin-AlexaFluor488 (ThermoFisher) was added per 100 µl PTwTx, and the embryos were stained overnight at 4°C. After eight 10-min washes with PTwTx, the embryos were gradually embedded in Vectashield (Vector labs) and imaged with the Leica SP8 CLSM.

Whole‐mount in situ hybridization of zebrafish embryos at 30 hpf was performed using a digoxigenin‐labeled antisense RNA probe. The pou4f1 cLone was provided by Steve Wilson UCL, UK. The probe that was specific for zic3 was synthesized from 1990 bp fragment cDNA using the DIG‐RNA labeling kit (Roche) according to the manufacturer's instructions. Whole‐mount in situ hybridization was performed as described previously53 with minor modifications. After rehydration, permeabilization, and blocking, the RNA probe was hybridized to target mRNA overnight at 68°C. Anti‐digoxigenin‐AP Fab fragments (Roche) were used for target recognition. The color reaction was performed using NBT/BCIP Solution (Roche).

We followed the methods described previously (Neugebauer et al., 2009 (link)) for immunostaining. The mixed immunofluorescence and in situ hybridization technique was adapted (Thisse and Thisse, 2008 (link)) as follows: on the second day, we added the antibody anti-GFP together with the antibody Anti-Digoxigenin-AP Fab Fragments (Roche) and incubated over night at 4°C in a horizontal rotator; on the third day, we added the secondary antibody anti-rabbit Alexa Fluor 488 and incubated over night at 4°C in a horizontal rotator; on the fourth day, we developed the RNA probe with Fast-Red substrate (Roche) until a red deposit was observed and the reaction was stopped with 4% PFA (in PBS) for 5 min. her12 RNA probe was a gift from Leonor Saude. Antibodies used were anti-acetylated α-tubulin (1:300; T7451 from Sigma), anti-GFP (1:500; ab290 from Abcam), anti-DlD (1:50; zdd2 monoclonal antibody [Itoh et al., 2003 (link)]), anti-mouse Alexa Fluor 564, and anti-rabbit Alexa Fluor 488 (1:500; Invitrogen). Nuclei were stained with DAPI (1:500). Flat-mounted embryos were examined with a Zeiss LSM 710 Meta confocal microscope and a Zeiss 40x water immersion C-Apochromat lens (1.2 NA). Three-colour confocal z-series images were acquired using sequential laser excitation, and analysed using Fiji software (LSM Reader) (Schindelin et al., 2012 (link)).

The ISH was conducted as described by Helmprobst et al. (2017) [48 (link)]. The testes were collected and fixed in 4% paraformaldehyde (PFA in PBS, pH 7.4) in the 4°C for 2 hours. After rinsing in PBS, the samples were dehydrated in 0.5 M sucrose (dissolved in PBS) in 4°C for another 2 hours, then embedded by Tissue-Tek O.C.T. Compound to store in the -40°C. For ISH, the samples were cut into 10 μm sections with Cryostat microtome. A length of 502 bp kifc1 fragment was cloned from cDNA and ligated to PGEM-T Vector with Sp6 and T7 transcription sites. Sense and antisense probe was transcribed with Sp6 and T7 RNA transcription enzymes (Promega, Madison, Wisconsin, USA) and the NTP DIG RNA labeling Mix (Roche, Basel, Switzerland), according to the Manufacturer's protocols. After hybridization with the DIG-RNA antisense probes, the slides were incubated with Anti-Digoxigenin-AP Fab fragments (Roche, Basel, Switzerland) for overnight, then stained with NBT/BCIP mix (Promega, Madison, Wisconsin, USA). The slides were then dehydrated in gradient alcohol as 50%, 70%, 90%, and 100% for 15 min each and permuted by xylene before sealed with neutral balsam. The sense probes were used as negative control. The pictures were taken by microscope (Olympus BX40). The bands were analyzed by software Image J.

To detect the expression of Meox-2 mRNA and Fgfr1 mRNA, we generated cRNA probes from the plasmids that contained full length cDNAs of Meox-2 and Fgfr1. The plasmid for Meox-2 has been described previously [12 (link)–14 (link)], and the plasmid for Fgfr1 was from Dr. Janet Rossant’s laboratory [15 (link)]. Whole mount in situ hybridization was carried out according to the protocol described by Shen [16 (link)]. Briefly, digoxigenin-labeled antisense RNA probes were hybridized, followed by incubation with anti-digoxigenin-AP Fab fragments (Roche), which can be detected by a color reaction using NBT/BCIP (Roche). For a given stage, at least 10 embryos of each strain were examined.

Chromogenic in situ hybridization ISH was carried out according to Dinhopl et al. [8 (link)]. In brief, 3 μm paraffin wax-embedded tissue sections were subjected to proteolytic treatment with proteinase K (Roche, Basel, Switzerland) 6 μg/ml in Tris-buffered saline at 37 °C for 50 min. For hybridization, the slides were incubated overnight at 40 °C with hybridization mixture and a final probe concentration of 100 ng/ml. The used oligonucleotide probe (sequence: 5′-TTTAATAACTCGTTATATATATCAGTGTAGCAC-3′) was labelled with digoxigenin at the 3′ end (Eurofins MWG Operon, Ebersberg, Germany). The probe is aimed at 18S rRNA strand and is specific to detect avian Plasmodium spp. [9 (link)]. The digoxigenin-labelled hybrids were detected by incubating the slides with antidigoxigenin-AP Fab fragments (Roche) (1:200) for 1 h at RT. Visualization of the reaction was carried out using the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 4-nitro blue tetrazolium chloride (NBT) (Roche). Probe specificity has been extensively tested previously [8 (link)]. Tissues from a deceased wild Blackbird Turdus merula free of avian malaria parasites as well as application of an irrelevant oligonucleotide probe (designed for Leishmania spp.) on the experimental samples [see 8 (link)] were used as controls in this study.