Partial cDNA fragments were amplified using primers specific for rat Xlr5c-like (XM_ 003748905, (5′-CAGACTTGAAAGAGGCCAGG-3′, 5′-TTTGCTGACTGCCAATGAAG-3′) and RT reactions of total RNA samples isolated from ovaries at 12 h post-hCG. The amplified PCR fragment was cloned into pCRII-TOPO Vector. Sequences of the cloned DNA were verified commercially (Eurofins Genomics). Plasmids containing partial cDNA for Xlr5c-like were linearized with HpaI and EcoRV to generate sense and antisense riboprobes, respectively. Linearlized plasmids were labeled using a fluorescein RNA labeling kit (Roche Applied Sciences) and T7 and SP6 RNA polymerase, as appropriate. Ovaries collected from immature rats injected with PMSG or PMSG + hCG were sectioned at 10 μm and mounted on Probe On Plus slides (Fisher Scientific). In situ hybridization analysis was carried out as described previously with a slight modification (Jo et al, 2004 (link)). Briefly, the ovarian sections hybridized with the probes were incubated with anti-fluorescein antibody (Roche Applied Sciences) at 4°C overnight and the signals for Xlr5c-like mRNA were amplified using a TSA™-plus fluorescein kit (Roche Applied Sciences). The sections were counterstained with propidium iodide for 20 min and fluorescent staining specific for Xlr5c-like mRNA was visualized with an Eclipse E800 Nikon microscope under fluorescent optics.
Fluorescein rna labeling kit
Manufactured by Roche
The Fluorescein RNA Labeling Kit is a product developed by Roche. It is used for labeling RNA molecules with fluorescein, a fluorescent dye. The kit provides the necessary reagents and protocols to facilitate the labeling process.
Automatically generated - may contain errors
Lab products found in correlation
Dig rna labeling kit, by Roche (5 mentions)
Nbt bcip, by Promega (2 mentions)
Ultrahyb ultrasensitive hybridization buffer, by Thermo Fisher Scientific (2 mentions)
Tsa plus dnp hrp system, by PerkinElmer (2 mentions)
Alkaline phosphatase conjugated anti fluorescein antibody, by Roche (2 mentions)
Hnpp fluorescent detection set, by Roche (2 mentions)
Probeon plus slides, by Thermo Fisher Scientific (1 mentions)
Anti fluorescein antibody, by Roche (1 mentions)
Eclipse e800 microscope, by Nikon (1 mentions)
Tsa plus fluorescein kit, by Roche (1 mentions)
Hrp conjugated anti dig antibody, by Roche (1 mentions)
Horseradish peroxidase hrp conjugated anti dig antibody, by Roche (1 mentions)
Anti fluorescein pod, by Roche (1 mentions)
Lsm 800 microscope, by Zeiss (1 mentions)
Tsatm plus fluorescein system, by PerkinElmer (1 mentions)
Leica tcs sp5 microscope, by Leica camera (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Anti digoxigenin pod, by Roche (1 mentions)
Tsa plus cyanine 3 cyanine 5 system, by PerkinElmer (1 mentions)
7 protocols using fluorescein rna labeling kit
1
In situ Hybridization of Rat Xlr5c-like
2
Whole-mount in situ hybridization protocol
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Single or double whole-mount in situ hybridizations were performed in glass vials as previously described (Paffett-Lugassy et al., 2013 (link)) or in 96-well mesh-bottom plates. Digoxygenin-labeled anti-sense RNA probes to nkx2.3, nkx2.5, tie1, etsrp, kdrl, tgfbr1a, tgfbr1b, tgfbr2a, tgfbr2b, tgfb1a, tgfb1b, tgfb2a, tgfb2b, tgfb3, and ltbp3 were synthesized using a DIG RNA Labeling Kit (SP6/T7/T3; Roche Applied Science). A blue (NBT/BCIP) chromogenic substrate was utilized (Promega). Double in situ hybridizations were performed using anti-sense fluorescein-labeled RNA probes to nkx2.3 that were synthesized using a Fluorescein RNA Labeling Kit (SP6/T7/T3; Roche Applied Science). An orange (INT/BCIP) chromogenic substrate was utilized (Roche Applied Science).
3
In Situ Hybridization of Zebrafish Opsin Genes
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Preparation of RNA probes and in situ hybridization were carried out as previously described (17 (link)). Digoxigenin (DIG)- and fluorescein-labeled antisense and sense RNA probes for zebrafish parapinopsin and parietopsin mRNAs were synthesized by using the DIG RNA labeling kit and fluorescein RNA labeling kit (Roche), respectively. Sections were pretreated with proteinase K and hybridized with each RNA probe in ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion). For double fluorescence labeling, sections hybridized with DIG-labeled probes were incubated with HRP-conjugated anti-DIG antibody (Roche) and subsequently treated with the TSA plus DNP (HRP) system (Perkin-Elmer), followed by incubation with Alexa 488-conjugated anti-DNP antibody. Fluorescein-labeled probes on the sections were detected by incubation with alkaline phosphatase-conjugated anti-fluorescein antibody (Roche) followed by a color reaction using the HNPP Fluorescent Detection Set (Roche).
4
Localization of Lamprey Photoreceptor Genes
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Preparation of the RNA probes and in situ hybridization were carried out as previously described [8 (link)]. Digoxigenin (DIG)- and fluorescein-labeled antisense and sense RNA probes for lamprey parapinopsin and parietopsin mRNAs were synthesized using the DIG RNA-labeling kit and fluorescein RNA-labeling kit (Roche), respectively. Sections were pre-treated with proteinase K and hybridized with each RNA probe in ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion). The pineal sections hybridized with DIG-labeled probes were incubated with horseradish peroxidase (HRP)-conjugated anti-DIG antibody (Roche) and subsequently treated with the TSA plus DNP (HRP) system (Perkin Elmer), followed by incubation with Alexa 488-conjugated anti-DNP antibody. The fluorescein-labeled probes were detected through incubation with alkaline phosphatase-conjugated anti-fluorescein antibody (Roche) followed by a color reaction using the HNPP Fluorescent Detection Set (Roche).
5
Dual-Label In Situ Hybridization of Germ Cell Markers
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Dual-label ISH protocol referred to our previous study [38 (link)]. The Vasa probe was 1040 bp and synthesized using Fluorescein RNA labeling kit (Roche). Gonadal sections were pre-hybridized for 2 h, then hybridized by 1 µg/mL Vasa probe and one of EcPou5f1 and EcNanog probes at the same time at 65 °C for 15 h. Subsequently, sections were washed with SSC and blocked with Blocking Reagent for at least 1 h. The Vasa probe was incubated with anti-Fluorescein-POD (Roche), then stained red fluorescence for 10 min with TSATM PLUS Fluorescein system (PerkinElmer, Shelton, USA). After being washed, the EcPou5f1 or EcNanog probes were incubated with anti-Digoxigenin-POD (Roche) and were stained green fluorescence for 10 min. Finally, gonadal sections were counterstained by DAPI (Solarbio) for cell nuclei staining. Sections were imaged with a Zeiss LSM 800 microscope (Zeiss, Jena, Germany) or a Leica TCS SP5 microscope (Leica).
6
Whole-mount in situ hybridization protocol
7
In vitro RNA Probe Synthesis and FISH
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Probes for ncoa5 and dmrt1 antisense/sense digoxigenin-labeled RNA strands were transcribed in vitro using the DIG RNA labeling kit (Roche). Probes for roar, cyp19a1a, and sf-1 antisense/sense fluorescein-labeled RNA strands were transcribed in vitro using the Fluorescein RNA labeling kit (Roche). Specific primers with a T7 RNA polymerase promoter were designed to amplify complementary DNA (cDNA) fragment of each gene (S2 Table ). Each probe was used at a final concentration of 0.5ng/μL. For more sensitive fluorescence in situ hybridization detection, the tyramide signal amplification TSA Plus Cyanine 3/Cyanine 5 System (PerkinElmer Life Science) was used according to the manufacturer’s instructions. Digoxigenin-labeled RNA was stained with cy5, fluorescein -labeled RNA was stained with cy3. FISH analyses using sense RNA strands were shown in S12 Fig .
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