Digoxygenin

Uninjured (n = 3) and injured (4 weeks post-injury; n
= 3) wild-type mice were deeply anaesthetized with sodium pentobarbital
(Euthatal: 80 mg/kg, i.p) and transcardially perfused with DPEC-treated sterile PBS
followed by 4% paraformaldehyde in 0.1 M phosphate buffer, and spinal cords were
harvested and prepared for immunohistochemistry. Tissue was post-fixed with 4%
paraformaldehyde in 0.1 M PBS for 3 h at 4 °C, cryoprotected for 24 h at 4 °C in
30% sucrose in diethyl pyrocarbonate-treated PBS prior to embedding and sectioned
at 30-µm thickness. Pan-Nrg1 in situ probes (Meyer et al., 1997 (link)) were transcribed in
vitro and labelled with digoxygenin (DIG) according to manufacturer’s
instructions (Roche). Following overnight hybridization at 65 °C, sections were
incubated with anti-DIG antibodies (Roche) and developed as previously described (Hopman et al., 1998 (link)). To
examine Nrg1 expression in different cell types, slides were then co-stained for P0, Olig2
and NeuN immunohistochemically, as described above.

Before cryosectioning, the eyes of cave and surface fish were stored in 30% sucrose at 4 °C overnight. The sections were stained with hematoxylin and eosin (H&E). For ISH, RNA probes were generated using Roche digoxygenin from Sinocyclocheilus cDNAs. The following primers were used to clone otx5 probes in PCR reactions: otx5-F: 5′-TGTGGTTTAAGAACCGTCGTG-3′ and otx5-R: 5′-GAACTTCCAGGAGTTCTGGTC-3′, which amplifies the exon3 of otx5. PCR amplification products were recovered from the gels with a DNA purification kit according to the manufacturer’s instructions (DP209; Tiangen Biotech) and then cloned into the pGM-T vector (VT202; Tiangen Biotech) in E. coli DH5a. Six clones were sequenced for every sample using T7 and Sp6 universal sequencing primers. Sequence-verified clones were used to generate antisense probes using SP6/T7 enzymes. ISH was performed with the color visualized using NBT/BCIP as described previously (Meng et al. 2013b (link)).

Whole mount in situ hybridization was carried out using standard protocols [21 (link)]. For preparation of antisense apoCI RNA probe, pBluescript SK+ 3–2 plasmid vector was linearized with Not I and transcribed in vitro with T7 RNA polymerase. For double in situ hybridization, probes were synthesized using digoxygenin or fluorescein RNA labeling mix (Roche) and detected with corresponding antibodies (Roche). First antibody reactions were quenched using 0.1M glycine-HCl, pH2.2 [22 (link)]. Magenta phosphate (Sigma), BCIP (Roche) and BM Purple (Roche) were used for chromogenic reactions. Other probes used in this study were Otx-A, Pax-6 and Slug [23 (link), 24 (link)]. 40 μM vibratome sections were obtained by embedding 4% paraformaldehyde-fixed embryos in 20% type B Bovine Gelatin (Sigma).

In situ hybridization and immunohistochemistry were performed as described previously (Schulte-Merker, 2002 ). Templates for in vitro transcription of enpp1 and spp1 were generated from cDNA. For enpp1, a combination of two probes was used for improved detection. Primer sequences are shown in supplementary material Table S1. Antisense digoxygenin-labeled mRNA probes were generated according to standard protocol (Promega SP6, RNA polymerase). digoxygenin was purchased from Roche.
For detection of YFP on embryos that had been stained for Trap, a rabbit antibody against GFP (1:300, Torrey Pines TP401) and an Alexa-Fluor-488-conjugated antibody against rabbit IgG were used (1:500, Molecular Probes A11034). Embryos were fixed in 4% paraformaldehyde and permeabilized with proteinase K (Promega, 15 μg/ml) for 3 minutes.

Digoxygenin (Roche) labelled RNA probes were made using standard protocols and spanned a minimum of 800 bp. To enhance permeabilisation, fixed embryos or larvae were dehydrated in methanol for a minimum of one hour at −20°C, rehydrated in PBST (PBS with 0.5% Tween-20, Sigma) and treated with 0.02 mg/ml proteinase K (PK, Sigma) for 10–40 min depending on the age of the fish. Probe hybridisation was carried out at 70°C in standard hybridisation solution containing 50% formamide over-night, with 2 ng/μl of RNA probe. Embryos were washed at 70°C through a graded series of hybridisation solution and 2x saline sodium citrate (SSC), followed by further washes with 0.2x SSC and PBST at room temperature. Blocking was carried out in maleic acid buffer (150 mM maleic acid, 100 mM NaCl, 2% sheep serum, 2 mg/ml BSA) for 2–3 hr. Probes were detected by over-night incubation with anti-Digoxigenin-AP Fab fragments (1:5000) (Roche) and stained with standard Nitro Blue Tetrazolium (NBT) and 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche) ISH protocol. Fluorescent in situ hybridisation (FISH) was carried out using either Fast Red tablets according to manufacturer’s instructions (Roche, discontinued from manufacturing) or Fast Blue BB Salt (Sigma) and NAMP (Sigma) staining as previously described (Lauter et al., 2011 (link)).

WISH was performed as described in our previous study [19 (link)]. Gene-specific primers were designed based on available information, and the PCR products were cloned into pGEM-T Easy vectors for anti-sense RNA probe synthesis. The primers are listed in Table S1. The anti-sense RNA probes labeled with digoxygenin (Roche Diagnostics) were used to verify the expression of target genes in whole-mount embryos. The images were taken under a Leica microscope (Leica M205FA, Germany).

Chromosome numbers and karyotypes were analysed as described by Jang et al. [15 (link),20 (link)] using standard Feulgen staining. Chromosomal spreads for FISH and GISH were prepared by enzymatic digestion and squashing, as described in Jang et al. [15 (link),20 (link)].
Probes used for FISH were: satellite DNA PaB6 isolated from the B⁶ genome in plasmid pGEM-T easy [28 (link)], 35S rDNA (18S/25S rDNA) from Arabidopsis thaliana in plasmid pSK+, and 5S rDNA from Melampodium montanum (Asteraceae) in plasmid pGEM-T easy, labeled with biotin or digoxygenin (Roche, Vienna, Austria). Probes were labeled either directly by PCR (5S rDNA and satellite DNA PaB6) or using a nick translation kit (35S rDNA; Roche). A commercially available, directly Cy3-labelled PNA (peptide nucleic acid) probe to vertebrate telomeric sequences (CCCTAA)₃ (Dako, Glostrup, Denmark) was used as described in [10 (link)]. FISH was performed as described in Jang et al. [15 (link),20 (link)].