Five micrograms of total RNAs were separated on 1% agarose gel containing 2% formaldehyde, and gels were treated with 0.05 N NaOH for 20 min to enhance transfer of high molecular weight RNAs, which step was essential to detect mNeat1_2/nNEAT1_2. RNAs were transferred on a positively charged Nylon membrane (Merck #11209299001) using standard protocol, and probe hybridization and detection were performed using DIG Easy Hyb (Merck #11603558001), anti-Dig AP (Merck #11093274910), and CSPD-Star (Merck #11685627001) following manufacturer's instructions. Chemiluminescence signals were detected with the ChemiDoc Touch Imaging System (Bio-Rad).
Anti dig ap
Manufactured by Merck Group
Anti-DIG-AP is a laboratory reagent used in detection and quantification assays. It is an antibody conjugated with alkaline phosphatase enzyme that specifically binds to digoxigenin (DIG), a small molecule commonly used to label nucleic acids. The core function of Anti-DIG-AP is to provide a sensitive, enzymatic signal for the detection of DIG-labeled targets in various analytical techniques.
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Lab products found in correlation
Dig easy hyb, by Merck Group (2 mentions)
Spei hf, by New England Biolabs (2 mentions)
Pcr dig labeling mix, by Roche (2 mentions)
Mlui hf, by New England Biolabs (2 mentions)
Blocking reagent, by Merck Group (2 mentions)
Bcip nbt, by Merck Group (2 mentions)
Nbt bcip, by Roche (2 mentions)
Anti dig pod, by Merck Group (2 mentions)
Nylon membrane, by Merck Group (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Genescreen plus, by PerkinElmer (1 mentions)
Genescreen plus hybridization transfer membrane, by PerkinElmer (1 mentions)
Fluorescein 12 utp, by Roche (1 mentions)
Fluorescein labeled pna, by Vector Laboratories (1 mentions)
Anti dnp hrp, by Vector Laboratories (1 mentions)
7 protocols using anti dig ap
1
Northern Blot for lncRNA Detection
2
Sensitive Neat1_2 Transcript Detection
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Five micrograms of total RNA was separated on a denaturing gel (1% agarose gel, 1× MOPS, 2% formaldehyde), the gels were rinsed twice in distilled water for 15 min, and the RNA samples were hydrolyzed in 0.05 N NaOH for 20 min. This alkali treatment step is critical for detecting Neat1_2 transcripts. After the samples were soaked in water and subsequently incubated twice in 20× SSC for 45 min, the RNA samples were transferred to a positively charged nylon membrane (Merck, #11209299001) and were hybridized with probes at a concentration of 1 µg/mL in DIG Easy Hyb (Merck, #11603558001). The hybridized probes were detected with anti-Dig AP (Merck, #11093274910) and CDP-Star (Merck, #11685627001) following the manufacturer's instructions.
3
Southern Blot Analysis of Transgenic Cowpea
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Genomic DNA of 10 µg from the cowpea lines of the cassettes AtRps5a pro Cre and AtUbq3 pro lox was digested overnight by SpeI-HF ® and MluI-HF ® (New England Biolabs), respectively. Restriction fragments were separated by gel electrophoresis and then transferred to GeneScreen Plus ® hybridization transfer membrane (Cat# NEF1017001PK, PerkinElmer, Waltham, MA, USA). Hybridization was conducted using a digoxigenin (DIG)-labeled probe targeting either the Cre gene for the cassette AtRps5a pro Cre or the ZsGreen gene for the cassette AtUbq3 pro lox and using DIG Easy Hyb TM as hybridization buffer following the Roche DIG application manual for lter hybridization (Eisel et al. 2008 ). The probes were generated and labeled by PCR with primers p3769/p3770 and p3700/p3701 (Table S3) for Cre and ZsGreen, respectively using the Roche PCR DIG labeling mix following the manufacturer's protocol. Detection was conducted using the chromogenic assay with NBT/BCIP according to the Roche DIG application manual for lter hybridization (Eisel et al. 2008) . All chemicals used for making buffers, as well as blocking reagent (Cat# 11096176001), anti-DIG-AP (Cat# 11093274910), and NBT/BCIP (Cat# 11681451001), were from Millipore Sigma.
4
Southern Blot Analysis of Cowpea Transgenics
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Genomic DNA of 10 µg from the cowpea lines of the cassettes AtRps5aproCre and AtUbq3prolox was digested overnight by SpeI-HF® and MluI-HF® (New England Biolabs), respectively. Restriction fragments were separated by gel electrophoresis and then transferred to GeneScreen Plus® hybridization transfer membrane (Cat# NEF1017001PK, PerkinElmer, Waltham, MA, USA). Hybridization was conducted using a digoxigenin (DIG)-labeled probe targeting either the Cre gene for the cassette AtRps5aproCre or the ZsGreen gene for the cassette AtUbq3prolox and using DIG Easy HybTM as hybridization buffer following the Roche DIG application manual for filter hybridization (Eisel et al. 2008 ). The probes were generated and labeled by PCR with primers p3769/p3770 and p3700/p3701 (Table S3) for Cre and ZsGreen, respectively using the Roche PCR DIG labeling mix following the manufacturer’s protocol. Detection was conducted using the chromogenic assay with NBT/BCIP according to the Roche DIG application manual for filter hybridization (Eisel et al. 2008 ). All chemicals used for making buffers, as well as blocking reagent (Cat# 11096176001), anti-DIG-AP (Cat# 11093274910), and NBT/BCIP (Cat# 11681451001), were from Millipore Sigma.
5
Whole-mount in situ hybridization protocol
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The probe template was generated using the following primers: Forward: 5’- TGCAGAACAGTCGACAAGGA-3’ and Reverse: 5’- GCTGGGCAATGCAATCGTAGC-3’. DIG-labeled antisense and sense probes were synthesized using T7 and SP6 RNA polymerase following the instructions provided in the MGEA script kit from Applied Bio. For hybridization, the eggs were dechorionated in 4% sodium hypochlorite for 1 min, fixed for 20 min in a 1:1 mixture of heptane and 4% paraformaldehyde in PBS, and devitellinized in 1:1 methanol/ heptane mixture and washed in PBST. In situ hybridizations on whole-mount fixed eggs were essentially performed as described before (Tautz, D. & Pfeifle, C, 1989). Then the embryos were blocked and incubated with primary antibody (anti-DIG-AP, Millipore Sigma Cat#11093274910) for 2 hours at RT. For color development reaction, the embryos were incubated in NBT/BCIP solution (Millipore Sigma, Cat#11681451001) at RT in the dark. The reaction was stopped by washing several times in PBS with 0.1% Tween 20. Embryos were photographed using a Nikon 90i Eclipse microscope with S Fluor 10x DIC N1 optics and a motorized stage.
6
In Situ Hybridization Protocols for Schistosoma
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FISH and WISH were performed as described previously (6 (link), 7 (link), 11 (link), 63 (link), 64 (link)). Parasites were fixed with 4% formaldehyde for 4 h (adults), or 4% formaldehyde containing 1% Nonidet P-40 and 0.2% Triton X-100 for 0.5 to 1 h (cercariae and schistosomula) or 1 to 2 h (juveniles), and then dehydrated in 100% methanol for storage. Five to 10 adult worms (male and female combined) and 5 to 15 juveniles and schistosomula were used for each gene analyzed in each biological replicate. Labeled FISH and WISH probes were generated using either DIG (digoxigenin)-12-UTP (Roche), or fluorescein-12-UTP (Roche). Probes were synthesized by in vitro transcription of partial gene sequences cloned in the plasmid vector pJC53.2, as previously described (63 (link)). Primers used for gene isolation by RT-PCR are listed in SI Appendix, Table S3 . Following probe hybridization, specimens were incubated with anti–DIG-AP (MilliporeSigma; 11093274910) for WISH, and anti-DIG-POD (MilliporeSigma; 11207733910) or anti–FITC-POD (MilliporeSigma; 11426346910) for FISH, between 1:1,000 and 1:2,000 dilution, and enzymatic labeling reactions carried out as previously described (64 (link)). Fluorescein-labeled PNA (Vector Labs) was used at 1:500 dilution in a FISH blocking solution overnight at 4 °C (65 (link)).
7
Colorimetric and Fluorescent In Situ Hybridization
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As previously described, colorimetric In situ hybridization (ISH) and fluorescent In situ hybridization (FISH) experiments were performed [31 (link)]. Specifically, 10–40 starved (at least four days) worms were killed and stripped of mucus by incubating them for 10 minutes in 7.5% (wt/vol) N-acetyl-L-cysteine (NAC) dissolved in PBS, followed by fixation in 4% (wt/vol) formaldehyde (Sigma-Aldrich #252549) in PBSTx (PBS + 0.3% Triton X-100, Fisher BioReagents, #BP151-500). Worms were stored in 100% methanol at -30°C for a minimum of 16h. Worms were bleached for 3 hours to overnight in formamide-containing solution under bright light, followed by incubation in a proteinase K solution (5 μg/mL proteinase K + 0.1% SDS in PBSTx). For colorimetric ISH, we used digoxigenin-containing (DIG) antisense probes in combination with anti-DIG-AP (alkaline phosphatase) antibody (1:2000, Millipore-Sigma #11093274910). For FISH we used DIG and/or DNP (dinitrophenol) containing probes, detected by tyramide signal amplification using anti-DIG-POD (peroxidase) (1:2000, Millipore-Sigma #11207733910) or anti-DNP-HRP (horseradish peroxidase) (1:2000, Vector laboratories #MB-0603). All samples in each experiment were processed in the same way in a side-by-side manner.
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