H7782
Manufactured by Merck Group
Sourced in Japan
The H7782 is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of this product is to facilitate standard laboratory tasks and procedures.
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4 protocols using h7782
1
Fluorescence In Situ Hybridization Analysis
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FISH analysis was performed as previously reported (Taguchi et al. 1993 (link)), with slight modifications. Metaphase preparations were denatured in 70% formamide/2x Saline-sodium citrate (SSC ) at 70 °C for 2 min; 0.8 μl of the prepared probe was mixed with 10 μl of hybridization solution (H7782, Sigma, Japan) and denatured at 82 °C for 10 min. Hybridization was performed at 37 °C in a CO2 incubator for 12–15 h. After hybridization, samples were washed twice in 2x SSC and 1x Phosphate buffered detergent (PBD; 0.05% Tween20/4xSSC). Chromosomes were counterstained with DAPI .
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2
In Situ Hybridization for miRNA-206 Detection
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The protocol for in situ hybridization for miRNA detection was based on a prior report.13 Specifically, 5‐μm sagittal lung sections were probed using a 3′‐fluorescein isothiocyanate–labeled miRCURY LNA rno‐miR‐206 detection probe (100 nmol/L; Exiqon, Vedbaek, Denmark). The miRCURY LNA U6 probe was used as a control. Probe (50 nmol/L LNA probe) annealing was performed in hybridization buffer (H7782; Sigma‐Aldrich, St Louis, MO) for 16 hours at 62°C. Following serial washes and inactivation of endogenous peroxide activity (3% hydrogen peroxide, 30 minutes at 25°C), immunolabeling was performed with an anti–3′‐fluorescein isothiocyanate horseradish peroxidase–conjugated secondary antibody for 1 hour at 25°C (1:100; DAKO, Carpinteria, CA). For detection, tyramide amplification was performed with the Individual Indirect Tyramide Reagent (PerkinElmer), and positive staining was evident by a green color. Nuclei were visualized with DAPI (Roche). The miR‐206 expression was quantified in the vascular wall of 10 to 15 resistance PAs (<100‐μm external diameter) in rats. Data are expressed as arbitrary units.
3
Telomere Visualization Protocol via Fluorescent In-Situ Hybridization
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A human digoxigenin-labeled telomere probe was purchased from Appligene Oncor (Lifescreen, Watford, UK). Random prime labeling of the probe DNA from PCR products was performed with FISH was conducted as previously reported (Takaoka et al. 2012 (link)), with slight modifications. In brief, the metaphase preparations were denatured in 70% formamide/2× saline-sodium citrate at 73 °C for 3 min. Then 1 μl of probe was mixed with 10 μl of hybridization solution (H7782, Sigma, Japan) and denatured at 80 °C for 10 min. HybridizaPageBreaktion of the probes was performed at 37 °C in the CO2 incubator for 12–15 h, followed by post-hybridization washes, DAPI counterstaining and visualization of the probes under a fluorescence microscope (Olympus BX50).
fluorescein-12-dUTP
cyanine-3-dUTP
(4’,6-diamidino-2-phenylindole)
4
Chromogenic Detection of Leishmania infantum
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Chromogenic in situ hybridization (CISH) was used to identify if the amastigote forms detected in the five positive cats for Leishmania spp. by IHC were those of L. infantum. We used CISH in order to associate tissue reaction with the presence of the parasite. For this purpose, a L. infantum-specific oligonucleotide probe labeled with digoxigenin that targets the minicircle kinetoplast DNA gene of the parasite was used (Menezes et al., 2013) (link). Five micrometer thick sections were cut from the paraffin blocks and mounted on silanized slides. These sections were processed as previously described (Boechat et al., 2016) (link), using the ZytoFastPlus chromogenic CISH Implementation Kit AP-NBT/ BCIP1 (Zytovision GmbH, Bremerhaven, Germany). The probe was diluted at 1:500 in the hybridization solution H7782 (Sigma-Aldrich Co., St. Louis, MO, USA). Positive and negative controls were included.
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