One-month-old juvenile fish (body length: 24.8 ± 2.3 mm) and embryos were utilized for the ISH and WISH experiments. The probes specific to SsMeox1 were amplified from cDNA using the primers listed in Table 1 . The ISH probes of the SsMeox1 gene were synthesized using a DIG RNA labelling kit (Roche, Mannheim, Germany). The ISH and WISH procedures followed previously established procedures [45 (link)]. The results of the ISH and WISH experiments were photographed using a Nikon AZ100 multizoom microscope (Nikon Corporation, Tokyo, Japan).
Dig rna labelling kit
Manufactured by Roche
Sourced in Germany, Switzerland, United States, China
The DIG RNA Labeling Kit is a laboratory tool for labeling RNA molecules with digoxigenin (DIG). The kit provides the necessary reagents and protocols for in vitro transcription of DIG-labeled RNA probes, which can be used for various applications such as Northern blotting, in situ hybridization, and RNase protection assays.
Automatically generated - may contain errors
Lab products found in correlation
Pgem t easy vector, by Promega (8 mentions)
Pcr purification kit, by Qiagen (4 mentions)
Nbt bcip, by Roche (4 mentions)
Alkaline phosphatase conjugated anti dig antibody, by Roche (4 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Dig nucleic acid detection kit, by Roche (3 mentions)
Pcr topoii, by Thermo Fisher Scientific (3 mentions)
Bx53 microscope, by Olympus (2 mentions)
Paraplast plus, by Leica (2 mentions)
T7 and sp6 polymerase, by New England Biolabs (2 mentions)
Pjet1, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Imagequant las 500 imager, by GE Healthcare (2 mentions)
Positively charged nylon membrane, by Roche (2 mentions)
Dig northern starter kit, by Roche (2 mentions)
T7 polymerase, by Roche (2 mentions)
Denhardt s solution, by Merck Group (2 mentions)
Blocking powder, by Roche (2 mentions)
Salmon sperm dna, by Merck Group (2 mentions)
92 protocols using dig rna labelling kit
1
In Situ Hybridization of SsMeox1 in Juvenile Fish
2
In-situ Hybridization Analysis of Polygalacturonase-like Genes in Fragaria vesca
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This study used Yellow Wonder 5AF7 (YW5AF7) seedlings, the 7th generation inbred lines of F. vesca. The plants were grown in a greenhouse (16 h/8 h light conditions at 22 °C, at a relative humidity of 65%) [26 (link)]. For cytological observation, the petal, stem, leaf, anther, filament, and fruit at 15 days after pollination (DAP), 20 DAP, and 25 DAP were sampled, then fixed in RNase-free FAA solution (4% formaldehyde, 50% ethanol, and 10% acetic acid). The fixed tissues were dehydrated in ethanol series and embedded in paraffin wax. After dewaxing, rehydration, sealing, and staining, the tissues were observed and recorded. Cross-section slicing (8 μm) was performed by Leica RM2255 (Leica Inc., Buffalo Grove, IL, USA).
A gene-specific cDNA fragment of FvePL1, 4, 7, 8, or 13 was individually amplified using ISH-F/R primer for in situ hybridization (Additional file1 ). Their PCR product was then cloned into the pGEM-T vector. A DIG RNA labelling Kit (Roche, German) was applied to the tissue paraffin Sect. [27 (link)]. Sense and antisense RNA probes were synthesized using SP6 and T7 RNA polymerase, respectively. In situ hybridization experiments were performed, including prehybridization, hybridization, washing, and detection [28 ]. Sides were photographed under a BX53 microscope (Olympus, Japan).
A gene-specific cDNA fragment of FvePL1, 4, 7, 8, or 13 was individually amplified using ISH-F/R primer for in situ hybridization (Additional file
3
Zebrafish Arginase II RNA Probe Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA probes for zebrafish arginase type II (arg2, ENSDARG00000039269; plasmid obtained from Source Bioscience) were designed and synthesised after cloning into the pCR Blunt II-TOPO vector, according to the manufacturer's instructions (Thermo Fisher Scientific). Plasmids were linearised and probes synthesised according to the DIG RNA Labelling Kit (SP6/T7) (Roche). Zebrafish larvae were fixed in 4% paraformaldehyde solution (Thermo Fisher Scientific) overnight at 4°C. Whole-mount in situ hybridisation was performed as previously described (Thisse and Thisse, 2008 (link)).
4
In Situ Hybridization of Barley Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Barley caryopses were fixed using FAA fixative (50% EtOH, 5% acetic acid, 10% formaldehyde) overnight in 4 °C, washed 2 times in 50% EtOH, dehydrated, and infiltrated with limonene, followed by Paraplast (Paraplast Plus, Leica) at 60 °C. The samples were embedded and cut into 12 µm thick transverse sections using a Histocore Autocut (Leica). Probe fragments were PCR-amplified using gene-specific primers (Supplemental Data Set S6 ) with preintegrated T3 or T7 promoter sequences. The probes were labeled with digoxigenin during in vitro transcription using a DIG RNA Labelling Kit (Roche) to produce sense and antisense riboprobes. The tissue samples were treated with proteinase K and hybridized with sense and antisense probes overnight at 55 °C. The digoxigenin incorporated into the probes was detected using an Anti-Digoxigenin-AP, Fab fragments (Roche) and a Vector Blue Alkaline Phosphatase (Blue AP) Substrate Kit (Vector Laboratories), and the sections were examined under a light microscope (Apotome Zeiss).
5
Whole-Mount In Situ Hybridization Protocol for Cestode Parasites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Digoxygenin (DIG)-labeled probes were synthesized by in vitro transcription with T7 and SP6 polymerase (New England Biolabs), using the DIG RNA labelling kit (Roche) according to the manufacturer’s instructions from emmekk1- and emmpk3-cDNA fragments cloned into vector pJET1.2 (Thermo Fisher Scientific). Primers for probe production are listed in S1 Table . The probes were subsequently purified using the RNEasy Mini Kit (Qiagen), analysed by electrophoresis, and quantified by dot blot serial dilutions with DIG-labeled control RNA (Roche). Whole-mount in situ hybridization (WISH) was subsequently carried out on in vitro cultivated metacestode vesicles essentially as previously described [2 (link),5 (link)], using vesicles of at least 1 cm in diameter to avoid losing material during washing steps. Fluorescent specimen were imaged using a Nikon A1 confocal microscope and maximum projections created using ImageJ as previously described [5 (link)]. In all cases, negative control sense probes yielded no staining results. In vitro labelling with 50 μM EdU was done for 5 hours (h) and fluorescent detection with Alexa Fluor 555 azide was performed after WMISH essentially as previously described [2 (link)].
6
Zebrafish Muscle Morphology Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to standard methods, 10-μm cryofrozen sections of 3-dpf-old larvae were stained with H&E and Gomori trichrome or subjected to immunohistochemistry analyses (Berger et al., 2010 (link)). Primary antibodies against dystrophin (1:20, Mandra1, DSHB), TMOD4 (1:200, 11753-1-AP, Proteintech) or actinin (1:1000, A7811, Sigma) were used; AlexaFluor-568-conjugated phalloidin was obtained from Life Technologies (1:1000, A12380). Fluorescence images were recorded using a Zeiss LSM 510 Meta fluorescence confocal microscope (Zeiss, Germany). For electron microscopy, 3-dpf-old larvae were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate overnight at 4°C. Electron micrographs of ultrathin sections were taken on a Hitachi H7500 transmission electron microscope (Hitachi, Japan). In situ hybridization was undertaken on whole mounts using a tmod4 digoxigenin-labelled antisense RNA probe that was synthesized using the DIG RNA labelling kit (Roche, Germany) and the plasmid IRBOp991E0136D (Source BioScience, UK) containing the tmod4 cDNA.
7
In-situ Hybridization of OsMS188 in Spikelets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The embedding block of wild type spikelets was sectioned to a thickness of 8 mm using an MR2 rotary microtome (RMC, USA). A 415-bp specific fragment of the OsMS188 CDS was cloned into a pBluescript-SK vector (Stratagene, USA). Plasmid DNA was completely digested using EcoRI or HindIII. The antisense and sense probes of OsMS188 were transcribed using a digoxigenin (DIG) RNA labelling kit (Roche, Switzerland) according to the product’s instructions. RNA hybridization and immunological detection of the hybridized probes were performed as described (Zhu et al. 2011 , Shi et al. 2018 (link)). Afterward, the samples were imaged via an Olympus DP73 digital camera (Olympus, Japan).
8
In Situ Hybridization Protocol for Embryonic Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-mount in situ hybridization was carried out as described previously (Hammes et al., 2001 (link)). In situ hybridization on sections was performed as described previously (Jensen and Wallace, 1997 (link)), except that the signal was enhanced by performing the colour reaction in the presence of 10% polyvinyl alcohol (Sigma Aldrich, P8136). Probe synthesis was conducted with the components of the DIG RNA labelling kit (Roche, 11277073910).
Images of embryos were taken using a Leica MZ 10F stereomicroscope (Leica LAS V4.9 imaging software). Images were processed in ImageJ to isolate specimen and adjust the background colour.
Primers used for cloning templates for in situ riboprobes: Lrp4 ISH probe primer forward: TAC CAT CGA AGC ATC TCG GC, reverse: TTC GTG TTT CCA GCC TGT GT; Lrp5 ISH probe primer forward: ATG CCG GCG GAG TGA AG, reverse: GAG TAG AAA GGC TCC CTC GG.
Lef1 riboprobe kindly provided by Thomas Willnow and Walter Birchmeier, MDC, Berlin, Germany.
Images of embryos were taken using a Leica MZ 10F stereomicroscope (Leica LAS V4.9 imaging software). Images were processed in ImageJ to isolate specimen and adjust the background colour.
Primers used for cloning templates for in situ riboprobes: Lrp4 ISH probe primer forward: TAC CAT CGA AGC ATC TCG GC, reverse: TTC GTG TTT CCA GCC TGT GT; Lrp5 ISH probe primer forward: ATG CCG GCG GAG TGA AG, reverse: GAG TAG AAA GGC TCC CTC GG.
Lef1 riboprobe kindly provided by Thomas Willnow and Walter Birchmeier, MDC, Berlin, Germany.
9
Northern Blot Analysis of RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Xcc cells were cultured and collected as described above. Total RNA was isolated using the PureLink RNA Mini kit (Thermo Fisher Scientific), and 3–5 μg of total RNA were separated on 6% denaturing (8 M urea) polyacrylamide gel and transferred to a positively charged nylon membrane (Roche Applied Science). After UV‐crosslinking, the membrane was hybridized with a DIG‐labelled RNA probe (prepared using a DIG RNA labelling kit [Roche Applied Science]) at 68 °C for 8 hr. Signal bands were detected using the DIG‐Northern Starter Kit (Roche Applied Science) and visualized with an ImageQuant LAS 500 imager (GE Healthcare). Signal bands were quantified using GelQuant.NET software (biochemlabsolutions.com).
10
In Situ Expression Analysis of Cosmos Flower Development
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A time series of capitula of both the wild-type and gh cosmos were collected at early developmental stages for in situ expression analysis. Tissues were fixed by dehydration in an increasing gradient series of ethanol, clearing with xylene, and embedding in Paraffin [36 (link),37 (link)]. The sections were cut in 8 µm (RM2265, Leica Biosystems, Germany).
Gene specific probes were synthesized for CbLFY (307 bp) and CbUFO(300 bp) according to Braissant and Wahli, 1998 [38 ], using a PCR-amplified fragment with a T7 promoter sequence appended to the 5’ end and labeled following the instructions of the DIG RNA labelling Kit (Roche, Basel, Switzerland). Hybridization and detection were carried out as described by Neta et al. [39 (link)]. Sections were examined and photographed using the BX53 Microscope (OLYMPUS) equipped with a U-HSCBM (OLYMPUS) digital camera.
Gene specific probes were synthesized for CbLFY (307 bp) and CbUFO(300 bp) according to Braissant and Wahli, 1998 [38 ], using a PCR-amplified fragment with a T7 promoter sequence appended to the 5’ end and labeled following the instructions of the DIG RNA labelling Kit (Roche, Basel, Switzerland). Hybridization and detection were carried out as described by Neta et al. [39 (link)]. Sections were examined and photographed using the BX53 Microscope (OLYMPUS) equipped with a U-HSCBM (OLYMPUS) digital camera.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!