Anti dig antibody conjugated to alkaline phosphatase

In situ hybridization (ISH) was performed as previously described (Gibson et al., 2013 (link); Liang et al., 2019 (link)). Briefly, the 5-µm tissue sections were processed in xylene and gradient ethanol for dewax and rehydration, followed by antigen retrieval with 10 μg/mL protease K (Ambion, Austin, TX) for 5 min at room temperature. The sections were then hybridized with 1 μg/mL antisense complementary RNA (cRNA) probe at 65 °C for 14–16 h. The probes used include 1.1 kb digoxigenin (DIG)-labeled DSPP cRNA probe and 0.8 kb DIG-labeled DMP1 cRNA probe. The sections were blocked and immunostained with an anti-DIG antibody conjugated to alkaline phosphatase (1:2000, Roche, Mannheim, Germany). The signals were developed with an NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) chromogenic substrate system (Roche ). The sections were counterstained with nuclear fast red (Sigma, Saint Louis, MO) and mounted with Permount mounting medium (Fisher Scientific, Waltham, MA). Images were taken using Leica DM4 B upright microscope (Leica Biosystems).

Probes for in situ hybridization were prepared as described in (29 (link)). In situ hybridization was performed as described in (30 (link)). For Digoxigenin labelled RNA probes synthesis, we used pBS-Srp (gift from C. Antoniewski). Briefly, ovaries were fixed for 30 min with 200 μl of 4% paraformaldehyde (PAF) in PBS, 20 μl of DMSO and 600 μl of heptane. They were washed in PBS Tween 0.1% (PBT), digested 20 min with 0.05 mg/ml proteinase K (in 50 mM Tris–HCl pH 7.5 5 mM EDTA), post-fixed 20 min with 4% PAF in PBS, dehydrated 1 h at −20°C in methanol/DMSO (9/1) and progressively rehydrated in PBT. They were pre-incubated for 1 h at 65°C in HB buffer (50% formamide, 2× SSC, 1 mg/ml Torula RNA, 0.05 mg/ml Heparin, 2% Roche blocking reagent, 0.1% CHAPS, 5 mM EDTA, 0.1% Tween 20) and incubated overnight with anti-sense DIG-labeled RNA probes. The samples were washed in HB for 1 h at 65°C, in HB/PBT (50/50) for 20 min at 65°C and several times in PBT at room temperature. Ovaries were incubated for 30 min in PBT–1% BSA before being incubated with anti-DIG antibody conjugated to alkaline phosphatase (Roche, 1:2000 in PBT–BSA) for 2 h. After several washes in PBT, in situ hybridization signals were revealed with FastRed (Roche) in Staining Buffer (100 mM NaCl, 50 mM MgCl₂, 100 mM Tris–HCl pH 8.2 an 0.1% Tween 20).

Whole-mount in situ hybridization was done as previously described [28] (link). Digoxigenin (DIG)-labeled sense and antisense RNA probes were made by in vitro transcription with T7 or SP6 RNA polymerase (Roche Diagnostics GmbH, Mannheim, Germany), using templates generated by PCR. Embryos were fixed in 4% paraformaldehyde (PFA) and stored in methanol. Rehydrated embryos were sequentially treated with Proteinase K and then acetylated in acetic anhydrate solution. The pretreated samples were next hybridized at 60°C with DIG-labeled RNA probe. The probe was detected with an anti-DIG antibody-conjugated to alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany) and visualized by using a BCIP/NBT solution kit (Nacalai Tesque Inc.).

Total cell RNA and mitochondrial RNA were extracted using the TRIzol^® method followed by column purification (Direct-zol™ RNA MiniPrep; Zymo Research, Irvine, CA). mRNA was further purified by polyA selection using a Dynabeads^® mRNA Purification Kit (Thermo Scientific, Waltham, MA). The RNA concentration and purity were checked using a NanoDrop™ (Thermo Scientific) and agarose gel electrophoresis. Then, 130 ng of each RNA sample was separated on a 10% precast polyacrylamide tris-borate-EDTA(TBE)-urea gel (Bio-Rad, Hercules, CA) and transferred onto a positively charged nylon transfer membrane (Ambion^®; Life Technologies). The blot was hybridized with 200 ng/mL digoxigenin (DIG)-labeled SHLP6 RNA probe in Easy Hyb™ hybridization buffer (Roche) for 16 h at 60°C. After two stringent washes in 2× SSC (saline sodium citrate buffer) (1× SSC contains 0.15 M NaCl and 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS) for 5 min at room temperature and 0.2× SSC-0.1% SDS for 15 min at 60°C, the membrane was incubated for 30 min at room temperature with an anti-DIG antibody conjugated to alkaline phosphatase (Roche). Subsequent visualization was achieved using the BCIP-NBT substrate as instructed using a nucleic acid detection kit (Roche).

The protocol for ISH is a modified method described elsewhere in detail^{58 (link),59 (link)}. Briefly: Collected tissues were fixed in 4% phosphate-buffered PFA for 24 h following incubation in 30% sucrose in PBS overnight. Afterwards samples were embedded in OCT and frozen at −20 °C. Blocks were sectioned at a cryotome with 15 µm thickness. Received sections were fixed on superfrost glass slides for 1 h at 55 °C. To detect Math6 mRNA expression by nonradioactive ISH, the digoxigenin-labeled specific anti-sense probe was produced by in vitro transcription with a DIG RNA labeling kit (Roche). The template is a 775 bp cDNA fragment of Math6 cloned into the pDrive vector (Qiagen). Permeabilization of the tissue was performed with proteinase K (10 µg/ml) for 20 min at room temperature. Afterwards 1 µg/µl probe was hybridized for 15 h at 65 °C. The hybridized probe was colorimetrically detected by an anti-DIG antibody conjugated to alkaline phosphatase (Roche).

Embryos at E7.5 were dissected from the uteri and isolated from decidua in RNase-free PBS. Then the embryos were fixed in 4% paraformaldehyde overnight at 4 °C and dehydrated with different concentration gradients of methanol (25%, 50%, and 75%, diluted with PBST: PBS containing 0.1% Tween-20) and stored in 100% methanol at −20 °C. Rehydrated embryos were prehybridized with a prehybridization solution at 70 °C for 3 h and then hybridized with digoxigenin-labeled riboprobes, which were generated by in vitro transcription from a linearized temple using a DIG RNA Labelling Kit (Roche, Cat#11175025910), at 70 °C overnight. Then embryos were blocked with a blocking solution containing Anti-DIG antibody conjugated to alkaline phosphatase (Roche, Cat#11093274910) overnight at 4 °C. The embryos were stained in 20 μl/ml NBT/BCIP (Roche, Cat#11681451001) at room temperature and then soaked in 80% glycerine until they sank to the bottom. Embryos were photographed on an OLYMPUS SZX16 microscope.