Antisense riboprobes that hybridize the intron 1 and 2 of the nascent RNA of Shh were synthesized as previously reported53 (link). Briefly, two plasmid vectors containing the intron 1 and 2 of Shh were linearized with NotI and SpeI, respectively. RNA labeling was performed with digoxigenin RNA labeling mix (Roche) by in vitro transcription with T3 and T7 RNA polymerases (promega), respectively. The fragment size of the riboprobes was reduced to 500 bp or fewer by alkaline hydrolysis. Four micrometers paraffin sections of mouse embryos were deparaffinized in xylene. The sections were rehydrated and treated with 1 μg/ml Proteinase K. After dehydration through ethanol series, they were hybridized with riboprobes overnight at 65 °C. Monoclonal anti-Digoxigenin (Sigma) and Alexa488-conjugated anti-mouse IgG (Thermo Fisher Scientific) were used as primary and second antibodies. Nuclei were stained with 1 μg/ml of DAPI (Thermo Fisher Scientific).
Digoxigenin rna labeling mix
Manufactured by Roche
Sourced in Switzerland
Digoxigenin RNA labeling mix is a reagent used in molecular biology applications to label RNA for detection and analysis. It contains digoxigenin-labeled nucleotides that can be incorporated into RNA through in vitro transcription or reverse transcription processes. The labeled RNA can then be detected using anti-digoxigenin antibodies or other detection methods.
Automatically generated - may contain errors
Lab products found in correlation
T7 rna polymerase, by Roche (4 mentions)
T3 or t7 rna polymerase, by Promega (2 mentions)
Szx16 microscope, by Olympus (2 mentions)
Dp80 ccd, by Olympus (2 mentions)
Sp6 rna polymerase, by Roche (2 mentions)
Tsa plus cyanine 5 kit, by Akoya Biosciences (2 mentions)
T7 polymerase, by Thermo Fisher Scientific (2 mentions)
Alexa 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
T3 polymerase, by Thermo Fisher Scientific (1 mentions)
Rq1 dnase, by Promega (1 mentions)
Ds ri1, by Nikon (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Maxiscript, by Thermo Fisher Scientific (1 mentions)
Nbt bcip substrate, by Roche (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
M165 stereoscope, by Leica (1 mentions)
23 protocols using digoxigenin rna labeling mix
1
In Situ Hybridization of Shh Introns
2
Gene Expression Analysis in Zebrafish
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Scl, cmyb, mpx, dhx15, gata1, and hbae1 were cloned from zebrafish cDNA and inserted to pGEM T Easy Vector (Promega) for antisense RNA synthesis. Probe plasmids lmo2, pu.1, lyz, dab2, and ephrinb2 were obtained from Tao Peter Zhong. Rag1 probe plasmid was a kind gift from Feng Liu. Antisense RNA probes were transcribed in vitro by T3, T7, or Sp6 polymerase (Sigma‐Aldrich) with Digoxigenin RNA Labeling Mix (Roche). Primers for probe plasmid construction are listed in Table S1 .
WISH was carried out according to the standard protocol.26 (link)
WISH was carried out according to the standard protocol.26 (link)
3
In situ Hybridization of ap-AKH in A. californica
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A 243-nt antisense digoxigenin-labeled riboprobe was generated from the full-length cDNA of ap-AKH using the digoxigenin RNA labeling mix (Roche, Indianapolis, MN). The riboprobe was used at 200 ng/ml in a standard colorimetric ISH and visualized with nitro-blue-tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate as previously described [29] (link). All sections were counterstained with methyl green, and neurons positive for ap-AKH were counted as described above for counting ap-AKH-ir neurons. ISH was performed on three separate A. californica, and each time yielded similar results.
4
Fluorescence in situ Hybridization for NPM
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The fluorescence in situ hybridization experiments were performed57 (link) using a DNA fragment of ~500 bp corresponding to the coding region of mouse NPM. The fragment was amplified by PCR using the following primers fused to either a T7 or T3 minimal promoter sequence: NPM forward, 5′-TAA TAC GAC TCA CTA TAG GGA CGG TTG AAG TGT GGT TCA G -3′, and NPM reverse, 5′-AAT TAA CCC TCA CTA AAG GAA CTT GGC TTC CAC TTT GG -3′. The PCR product was used as the template for in vitro transcription of the NPM probe needed for fluorescence in situ hybridization. The antisense (T3) and sense (T7) probes were prepared using digoxigenin-RNA labeling mix (Roche Diagnostics). The RNA probes were quantified, denatured, and incubated with permeabilized cells57 (link). After the hybridization, the cells were used for immunofluorescence to detect HuR57 (link).
5
Zebrafish Caudal Fin Regeneration
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Zebrafish, 6-8 months of age, were used for caudal fin amputations. Fish were anaesthetized in tricaine and amputations were made by using a razor blade, removing half of the fin [27 (link)]. Animals were allowed to regenerate for 2 days in water kept at 31-33 °C; these temperatures facilitate more rapid regeneration than more commonly used temperatures of 25–28 °C [39 (link)]. Then, fish were anaesthetized, and the fin regenerate was removed for analyses. lef1 probes were transcribed in vitro by T3 polymerase (Ambion) with Digoxigenin RNA Labeling Mix (Roche). In situ hybridization of zebrafish fins was performed as previously described [40 (link)].
6
Circadian Rhythm Expression Profiling
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Larvae previously exposed to temperature or light cycles were fixed in 4% paraformaldehyde in PBS at different time points during 24 hours. In situ hybridization was carried out as previously described [31 ] using the zfaanat2 probe [20 ] that was labeled with digoxigenin RNA-labeling mix (Roche).
7
Synthesis and Validation of Sema3D/F Probes
8
RT-PCR and In Situ Hybridization for Gene Expression
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A fragment of each gene was amplified by RT-PCR using cDNA obtained from gonads as the template. The sequences of primers are listed in Table S6 . RNA extraction and cDNA synthesis were performed as described earlier. The CDS sequence of chicken NR5A1 was inserted in a pBluescript SK. The PCR products of other genes were subcloned using the pGEM T-Easy Vector System (Promega, Madison, WI, USA). cDNA clones were labelled using Digoxigenin RNA Labeling Mix (Roche, Basel, Switzerland) and T7, SP6 or T3 RNA polymerase (MAXIscript; Thermo Fisher Scientific). Hybridization to serial frozen sections was performed as described previously23 (link). The incubation temperature was modified to 65–70 °C. Images were captured using a cooled CCD camera (DS-Ri1; Nikon, Tokyo, Japan) mounted on a Nikon ECLIPSE E800 microscope and were analysed using NIS ELEMENTS (Nikon).
9
Whole Mount In Situ Hybridization Protocol
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Whole mount in situ hybridization was performed using standard procedures (Thisse and Thisse, 2008 (link)). The embryos were fixed in 4% PFA (Sigma Aldrich) and dehydrated in methanol before being hybridized with riboprobes that were generated from PCR products with the following primers: kdrl-fwd 5′-TGGCAGGATTCACTTTGAGTGG-3′ and kdrl-rev 5′-TAATACGACTCACTATAGGGTAGTGTAGGGCTCAATCCGCAG-3′; dlg4a-fwd: 5′-CGGCTACACATGAACAAGCAGC-3′ and dlg4a-rev: 5′-TAATACGACTCACTATAGGGGACGCTCCCTGGTGGGAATCC-3′; dlg4b-fwd: 5′-CATATGGACATGTCTGATTACCCG-3′ and dlg4b-rev: 5′-TAATACGACTCACTATAGGGACGGCCTGCGACAGAAAACC-3′; magi3a-fwd: 5′-CCTGCCAGCCGAGAAGACAGG-3′ and magi3a-rev: 5′-TAATACGACTCACTATAGGGCTTTCCAGGGACCTGGAGTTATGG-3′; magi3b-fwd: 5′-CGCTCAAGAGGAAGAAACACTGG-3′ and magi3b-rev: 5′-TAATACGACTCACTATAGGGTCCAACAGTAATCCACTCTCCTCC-3′
The probes were then transcribed with a T7 RNA polymerase (Roche) and digoxigenin-RNA labeling mix (Roche) and an anti-DIG AP (1:5000) and NBT/BCIP substrate (Roche) was used to detect them. The embryos were cleared with Benzyl Alcohol/Benzyl Benzoate (BA/BB) before being transferred into glycerol for storage and imaging using a Leica M165 stereoscope.
The probes were then transcribed with a T7 RNA polymerase (Roche) and digoxigenin-RNA labeling mix (Roche) and an anti-DIG AP (1:5000) and NBT/BCIP substrate (Roche) was used to detect them. The embryos were cleared with Benzyl Alcohol/Benzyl Benzoate (BA/BB) before being transferred into glycerol for storage and imaging using a Leica M165 stereoscope.
10
Mouse BDNF DNA Plasmid Characterization
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Mouse BDNF DNA plasmid (accession #X55573) was kindly donated by Dr. Stanley Watson, University of Michigan, Ann Arbor. The DNA sequence was validated by comparison to Bdnf reference sequence using FinchTV software (Geospiza, Inc., Seattle, WA, United States). Sense and antisense strands were generated by in vitro transcription with the AMBION Maxiscript T7 kit (Ambion, Waltham, MA, United States), labeled with digoxigenin RNA labeling mix (Roche, Basel, Switzerland), and purified with mini columns according to the manufacturer’s protocol. (Roche, Basel, Switzerland).
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