After immunohistochemical identification of the particular GCT-subtypes, the corresponding tumor sections were subsequently processed for in situ hybridization (ISH) with a miRCURY LNA probe (Exiqon, Vedbaek, Denmark; probe ID 38555-15) specific for miR-371a-3p. The protocol was performed according to the manufacturer’s instructions using a proteinase-K concentration of 15 μg/ml, a hybridization temperature of 51° C and a probe concentration of 80 nM. Microscopic evaluations were performed on an Axioskop 2 plus microscope (Zeiss, Göttingen, Germany). Histological findings were documented using the AxioCam HRc digital camera (Zeiss, Göttingen, Germany) and then edited with AxioVision Software v.4.8 (Zeiss, Göttingen, Germany). Presence of miR-371a-3p within GCTs was defined by distinct blue staining of the cells, and accordingly, only these cells were considered miR-371a-3p positive. Only the presence or absence of the miR-371a-3p in the specimen was evaluated, no quantification was attempted.
Mircury lna probe
Manufactured by Qiagen
Sourced in Denmark, United States
The MiRCURY LNA probes are a line of oligonucleotide-based hybridization probes designed for the detection and quantification of microRNAs (miRNAs) in various sample types. These probes utilize Locked Nucleic Acid (LNA) technology to provide enhanced affinity and specificity for target miRNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Nbt bcip, by Roche (4 mentions)
Bm purple ap substrate, by Roche (3 mentions)
Tyramide signal amplification system, by PerkinElmer (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Axiovision software v4, by Zeiss (2 mentions)
Axioskop 2 plus microscope, by Zeiss (2 mentions)
Axiocam hrc digital camera, by Zeiss (2 mentions)
Aqua poly mount, by Polysciences (2 mentions)
Torula yeast rna, by Merck Group (1 mentions)
Dig oligonucleotide 3 end labeling kit, by Roche (1 mentions)
Eclipse 800 microscope, by Nikon (1 mentions)
Mowiol mounting medium, by Merck Group (1 mentions)
Species specific secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Aperio scanscope xt, by Leica (1 mentions)
Alkaline phosphatase conjugated anti dig fab fragments, by Roche (1 mentions)
Blocking buffer, by Roche (1 mentions)
Positively charged nylon membrane, by Roche (1 mentions)
Ultrasensitive hybridization buffer, by Thermo Fisher Scientific (1 mentions)
Cdp star chemiluminescent substrate for alkaline phosphatase, by Roche (1 mentions)
19 protocols using mircury lna probe
1
In Situ Detection of miR-371a-3p in GCTs
2
LNA in situ Hybridization for miRNA Detection
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For the LNA in situ hybridization, miRCURY LNA probe was ordered from Exiqon (dme-miR-980 product # 21455-15). Tissues were dissected, fixed in 4% formaldehyde in PBS, dehydrated and stored at −20 °C overnight or longer. Tissues were then rehydrated, treated for 10 min with Proteinase K solution (50 μg/ml Proteinase K in 50 mM Tris–HCl pH 7.5, 50 mM EDTA), and post-fixed in 4% formaldehyde in PBT for 30 min. Tissues were prehybridized for 1 h in hybridization buffer (50% formamide, 25% 20 × SSC, 5 mg/ml Torula yeast RNA (Sigma), 0.1% Tween 20). Hybridization was carried out overnight at 60 °C with a 40 nM probe concentration in hybridization buffer. Post hybridization washing was done at 62 °C with three subsequent 20 min washes with hybridization wash solution (no yeast RNA), 50/50 v/v hybridization wash solution/PBT, PBT. Tissues were then blocked for 1 h in western Block (Sigma). Anti-DIG conjugated with Alkaline Phosphatase was diluted 1:2000 in block and incubated with tissues overnight at 4 °C. Colorimetric detection was done with 10 μl/ml NBT (Roche) in staining buffer (0.05 M Tris pH 9.5, 0.05 M MgCl2, 0.1 M NaCl, 0.1% Tween 20) for ~30–45 min.
3
In Situ Hybridization for miR-371a-3p in GCNis
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Sections with clear immunohistochemical evidence of GCNis were then processed with in situ hybridisation with a miRCURY LNA probe (Exiqon, Vedbaek, Denmark; probe ID 38555-15) specific for miR-371a-3p. The protocol was performed according to the manufacturer’s instructions using a proteinase K concentration of 15 µg/ml, a hybridisation temperature of 51 °C and a probe concentration of 80 nM. Microscopic evaluations were performed on an Axioskop 2 plus microscope (Zeiss, Göttingen, Germany) at 100× to 200× magnifications. Histological findings were documented using the AxioCam HRc digital camera (Zeiss, Göttingen, Germany) and then edited with AxioVision Software v.4.8 (Zeiss, Göttingen, Germany). Presence of miR-371a-3p within GCNis cells was defined by distinct blue staining of intratubular cells, and accordingly, only these cells were considered miR-371a-3p positive. Only the presence or absence of the miR-371a-3p in the specimen was evaluated, no quantification was attempted.
4
Localization of miR-183 in Zebrafish
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RNA probes against miR-183 were hybridized to fish fixed at 120 hpf. Hybridization detection was via alkaline phosphatase (AP)-conjugated anti-digoxigenin (Roche, Indianapolis, IN, USA), followed by the NBT/BCIP color reaction. Whole-mount in situ hybridization was performed using locked nucleic acid (LNA) probes for miR-183 labeled with digoxigenin (DIG Oligonucleotide 3′-End Labeling Kit; Roche). RNA probes for in situ hybridization were synthesized using Taq polymerase. LNA probes (miRCURY LNA probes) were purchased from Exiqon (Vedbaek, Denmark) or custom synthesized (Integrated DNA Technologies, Coralville, IA, USA) by incorporating LNA modifications at every third nucleotide position from the 5′ end. LNA probes are antisense to miR-183 (5′-CAGTGAATTCTACCAGTGCCATA). Briefly, fixed tissues were defatted with ethanol, digested with 10 µg/mL Proteinase K/PBT, hybridized with 12 pmol labeled LNA probe, washed, and digested with RNase A. Labeled LNA probe was detected using AP conjugated sheep anti-DIG Fab fragment and BM Purple AP Substrate (Roche). Tissues were whole mounted in glycerol and imaged using light microscopy using a Nikon Eclipse 800 micro-scope. A minimum of three samples were prepared for each condition.
5
Whole-mount in situ Hybridization and Immunostaining
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Whole-mount in situ hybridization (WISH) was performed as previously described (Pearson et al., 2009; (link)
Sasidharan et al., 2013; (link)
King and Newmark, 2013 (link)). miRNA-based WISH was carried out using digoxigenin-labeled miRCURY LNA probes obtained from Exiqon. For immunostaining, animals were fixed using Carnoy's solution as described (Sánchez Alvarado and Newmark, 1999 (link)). Rabbit anti-ARRESTIN (1:5000, clone VC-1; gift of Dr Kiyokazu Agata, Kyoto University, Japan) and mouse anti-SYNORF-1 (1:100, DHSB) primary antibodies were used. Species-specific secondary antibodies were obtained from Molecular Probes (1:400). Hoechst 33342 (25 µg/ml; Sigma) was used as a nuclear counterstain. Animals were mounted with Mowiol mounting medium (Sigma) and stored at 4°C until imaging.
Sasidharan et al., 2013; (link)
King and Newmark, 2013 (link)). miRNA-based WISH was carried out using digoxigenin-labeled miRCURY LNA probes obtained from Exiqon. For immunostaining, animals were fixed using Carnoy's solution as described (Sánchez Alvarado and Newmark, 1999 (link)). Rabbit anti-ARRESTIN (1:5000, clone VC-1; gift of Dr Kiyokazu Agata, Kyoto University, Japan) and mouse anti-SYNORF-1 (1:100, DHSB) primary antibodies were used. Species-specific secondary antibodies were obtained from Molecular Probes (1:400). Hoechst 33342 (25 µg/ml; Sigma) was used as a nuclear counterstain. Animals were mounted with Mowiol mounting medium (Sigma) and stored at 4°C until imaging.
6
In situ Hybridization for miRNA Detection
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In situ hybridization was performed on formalin-fixed paraffin-embedded sections (4 μm thickness) of tumor specimens. After processing with 3% H2O2, sections were treated with proteinase K (2 μg/ml) at 37°C for 30 min, washed, and prehybridized for 2 h at 37°C. Hybridization with digoxygenin (DIG)-labeled miRCURY LNA probes (probe sense: 5′-cACTTATCAGGTTGTATTATAa -3′; Exiqon, Woburn, MA, USA) was performed overnight at37°C. Slides were then washed at 37°C and incubated with alkaline phosphatase–conjugated sheep anti-DIG Fab fragments for 1h at room temperature. Staining was visualized by adding BM purple AP substrate (Roche, Basel, Switzerland) according to the manufacturer's instructions.
7
In Situ Hybridization of Mouse Embryos
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In situ hybridizations were performed on CD-1 mouse embryos with miRCURY LNA probes (Exiqon). For detailed protocol, see the Supplemental Material .
8
miRNA in situ Hybridization in FFPE Tissue
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miRNA in situ hybridization (ISH) was performed on FFPE tissue sections as previously described [15 ]. Briefly, a combination of double DIG‐conjugated mirCURY LNA probes (Exiqon, Vedbæk, Denmark) and an automatic DAB chromogenic detection system was used for detection of miRNAs. Probes selected for ISH analysis are listed in Table S1 . A scramble probe was used as negative control.
Samples were hybridized with probe mixture for 2 h, in the Dako Hybridizer, at specific probe hybridization temperature (RNA Tm −30 °C) (TableS1 ). After washing steps, miRNA expression was automated detected with the Ventana BenchMark ULTRA instrument using the OptiviewDAB Detection Kit (Ventana Medical Systems Arizona, Oro Valley, AZ, USA). For image analysis, stained sections were scanned with Aperio ScanScope XT (Leica Biosystems, Nussloch, Germany) and miRNAs signals were quantified as number of positive cells/total cells by counting three random field for each slide.
Samples were hybridized with probe mixture for 2 h, in the Dako Hybridizer, at specific probe hybridization temperature (RNA Tm −30 °C) (Table
9
In situ Hybridization of Nasopharyngitis and NPC
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In situ hybridization was performed on formalin-fixed, paraffin-embedded sections (5 μm) of nasopharyngitis and NPC tissue. Briefly, after dewaxing, sections were treated with proteinase K (2 μg/ml) at 37 °C for 15 min, washed and prehybridized for 1 h at 49 °C. Hybridization with digoxigenin (DIG)-labeled miRCURY LNA probes (Exiqon) was performed overnight at 49 °C. Slides were then washed at 49 °C and incubated with alkaline phosphatase-conjugated anti-DIG Fab fragments (1 : 1500; Roche Applied Science, Indianapolis, IN, USA) for 1 h at room temperature. The staining was visualized by adding BM purple AP substrate (Roche Applied Science) according to the manufacturer's instructions.
10
miR-203 Expression in Invasive CSCC
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In situ hybridization was carried out to determine the transcriptional level of miR-203 in the frozen skin tissue sections collected from patients with invasive CSCC. Briefly, the sections were hybridized with miRCURY LNA probes (Exiqon, Woburn, MA, USA) labeled with digoxygenin overnight, followed by incubation with alkaline phosphatase-conjugated antibody to digoxygenin at 4°C overnight. Alkaline phosphatase reaction was performed in polyvinyl alcohol (PVA) with MgCl2 and NBT/BCIP (Roche, Florence, SC, USA). Digoxygenin-labeled-locked nucleic acid miR scramble (Exiqon, Woburn, MA, USA) was used as controls for hybridization. Images were subsequently captured using Nikon Eclipse E400Q imaging camera (Nikon, Tokyo, Japan) and analyzed with the use of NIS-Elements’ BR 3.10 software.
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