Whole-mount in situ hybridization in zebrafish was performed as previous study 67 (link), 68 (link). Digoxigenin-labelled antisense RNA probes were produced with a DIG RNA labeling kit (Roche, #11175025910). The following probes were used (forward/reverse): trio (5'-TACCTGTCCACACACACCT-3'/5'-GGTACGATGAGATGGAAT-3'), foxd3 (5'-CAAAGCATGTGTCATCTTG-3'/5'-TGAGAATGTCCGGCTGAT-3'), crestin (5ʹ-TGCCCTGGAGACGAAACA-3ʹ/5ʹ-CCCACTTCCGATCTGCTT-3ʹ). The whole-mount in situ hybridization in mice was performed according to the protocol of the ISH kit (Boster Bio, #MK1031). The probe of trio was used (forward/reverse): 5'-GTCCTTAAGGCATCCAGTATC-3'/5'-CAAGGCCTCTTCAAGGTTATT-3'). Briefly, tissue samples were digested with pepsin, then incubated with pre-hybridization solution, followed by hybridizing with digoxigenin-labelled oligo-nucleotide probes overnight. On the next day, the samples were washed with SSC (NaCl+C6H5O7Na3·2 H2O) and blocked with blocking reagent at 37 ºC, then incubated with alkaline phosphatase-labelled anti-digoxin reagent. Finally, the samples were stained with BCIP/NBT and imaged by upright microscope (Leica Microsystems, Ontario, Canada).
Ish kit
Manufactured by Boster Bio
Sourced in China, United States
The ISH kit is a laboratory tool used to detect and locate specific DNA or RNA sequences within tissue samples. It utilizes in situ hybridization techniques to visualize the target genetic material.
Automatically generated - may contain errors
Lab products found in correlation
Dig rna labeling kit, by Roche (5 mentions)
Pannoramic viewer, by 3DHISTECH (3 mentions)
Pannoramic scan, by 3DHISTECH (2 mentions)
Dab kit, by Solarbio (2 mentions)
Upright microscope, by Leica (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Dig dutp, by Thermo Fisher Scientific (1 mentions)
Afap1 as1, by Thermo Fisher Scientific (1 mentions)
Nbt bcip solution, by Roche (1 mentions)
Anti digoxigenin alkaline phosphatase antibody, by Roche (1 mentions)
Ish probes, by Boster Bio (1 mentions)
Nbt bcip, by Roche (1 mentions)
Pannoramic scan instrument, by 3DHISTECH (1 mentions)
Ish probe, by Qiagen (1 mentions)
27 protocols using ish kit
1
Whole-mount in situ hybridization for gene expression analysis in zebrafish and mice
2
In Situ Hybridization for LINC01016, miR-302a-3p, and miR-3130-3p
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The ISH probe used for detecting LINC01016, miR-302a-3p, and miR-3130-3p labeled digoxin was designed and synthesized by Boster Bio-Engineering Company (Wuhan, China). The probe sequences were designed as LINC01016: 5′-TGTCACAGGCCAAGGGGATAGTTCACCACCTTGTTTTCTC-3′; miR-302a-3p: 5’-TCACCAAAACATGGAAGCACTTA-3’; miR-3130-3p: 5′-TTACCCAGTCTCCGGTGCAGC-3′. ISH was performed using the ISH Kit (Boster) according to the manufacturer’s instructions. Briefly, probes were diluted in hybridization buffer, denatured, and then hybridized overnight at 60 °C. The slides were blocked at 37 °C for 30 min and were visualized with 3,3'-diaminobenzidine reaction. Images were digitally acquired on a microscope.
3
SNHG12 Expression in NSCLC by ISH
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ISH was used to detect SNHG12 in 40 paired NSCLC and adjacent normal samples. A digoxigenin (DIG)-UTP-labeled antisense RNA probe (Beijing View Solid Biotechnology, Beijing, China) which was derived from nucleotides 28–257 of the SNHG12 coding sequence was obtained by in vitro transcription using a DIG RNA Labeling kit (Roche Diagnostics, Basel, Switzerland). The sense RNA probe derived from nucleotides 28–257 of the SNHG12 coding sequence was labeled with DIG-UTP and used as a negative control. ISH was performed using the ISH kit (Boster Biological Technology, Pleasanton, CA, USA), according to the manufacturer's protocol.
4
In Situ Hybridization of lncRNA Targets
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In situ hybridization (ISH) was performed by employing the ISH kit from Boster (Wuhan, China) as previously described. Cells in the clinical specimens (10 μm) were fixed and permeablized using xylenes, ethanol and protease to allow biotin-labeled probes to access. Slides were treated with 30% H2O2 and ddH2O with the ratio of 1: 10 for 5 min, and then the 3% citric acid diluted pepsase was applied to expose the fragment of nucleic acid for 20 s.The second fixation was followed by using 1% paraformaldehyde/0.1 M PBS. Next, the slides were incubated with pre-hybridization solution at 40 °C for 2 h and then with lncRNA target probes at 30 °C overnight followed by 2 washes with 2× saline sodium citrate (SSC). After blocking, biotin-labeled anti-digoxin was added and incubated for 60 min. Finally, slides were stained with DAB, dehydrated with 100% ethanol and xylene, and mounted in a xylene-based mounting media. The slides were recorded by Pannoramic SCAN (3D HISTECH, Budapest, Hungary) and analyzed by Pannoramic Viewer (3D HISTECH, Budapest, Hungary).
5
In Situ Hybridization of KCNQ1OT1 in Paraffin Tissue
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As per the directions of an ISH kit (BOSTER), paraffin-embedded tissue samples were utilized for ISH to assess KCNQ1OT1 expression. The paraffin slices were dewaxed and hydrated before 10-min immersion in xylene. The slices were soaked in xylene for another 10 min after changing xylene. The slices were positioned in 100%, 75%, and 50% alcohol for 5 min, and then in distilled water for 3 min. Following immobilizing, pre-hybridization, and hybridization, the slices were supplemented with biotinylated rat anti-digoxin and biotinylated peroxidase, and stained with a diaminobenzidine Kit (Solarbio) in the light of the instructions.
6
In-situ Hybridization for CASC11 in HCC
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The paraffin‐embedded HCC and peripheral liver samples were stained to detect CASC11 expression following the instructions of the in situ hybridization (ISH) Kit (BOSTER, Wuhan, China). In brief, the specimens were deparaffined, rehydrated, and then incubated with proteinase at 37°C for 10 min. After a three‐time wash in PBS, the specimens were incubated with hybridization mix at 40°C overnight. Afterward, the sections were blocked by incubation with blocking buffer for 30 min, and then were applied with anti‐DIG for 60 min. A DAB kit (Solarbio, Beijing, China) was used to finally stain the samples; the positive signal was indicated as blue. The probe sequences are listed in Table S3.
Besides, human HCC tissue microarray slides were acquired by purchase from US Biomax (Rockville, MD, USA) and were subjected to ISH staining as described above.
Besides, human HCC tissue microarray slides were acquired by purchase from US Biomax (Rockville, MD, USA) and were subjected to ISH staining as described above.
7
miR-148a Expression Analysis by ISH and IHC
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ISH was performed using the ISH Kit (Boster, Bio-Engineering Company, Wuhan, China). All procedures followed the manufacturer’s instructions. Samples were stained with hematoxylin, dehydrated with alcohol, washed with xylene, sealed with flavor sealing tablets. Oligo (5’Digoxin-ACAAAGTTCTGTAGTGCACTGA) was used as ISH probe for miR-148a. IHC staining was performed as described previously [24 (link)]. Primary antibody for SMAD2 (1:100, 12,570–1-AP) and Ki67 (1:100, 27,309–1-AP) were purchased from Proteintech. The representative images of ISH and IHC were captured and processed using DM2300 microscope and ScopeImage 9.0 software (Nanjing Jiangnan Novel Optics Co., Ltd., China). ISH and IHC staining scores were independently determined by 3 pathologists without prior knowledge of patient information. The overall score defined by multiplying the percentage of positive cells by the staining intensity score as described previously [24 (link)].
8
Comprehensive lncRNA In Situ Hybridization
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In Situ Hybridization was performed by employing the ISH kit from Boster (Wuhan, China) as previously described54 (link). Cells in the clinical specimens(10 μm) were fixed and permeablized using xylenes, ethanol and protease to allow biotin-labelled probes to access. Slides were treated with 30% H2O2 and ddH2O with the ratio of 1:10 for 5 min, and then the 3% citric acid diluted pepsase was applied to expose the fragment of nucleic acid for 20 s.The second fixation was followed by using 1% paraformaldehyde/0.1 M PBS. Next, the slides were incubated with pre-hybridization solution at 40 °C for 2 h and then with lncRNA target probes at 30 °C overnight followed by two washes with 2 × saline sodium citrate. After blocking, biotin-labelled anti-digoxin was added and incubated for 60 min. Finally, slides were stained with DAB, dehydrated with 100% ethanol and xylene, and mounted in a xylene-based mounting media. The slides were recorded by Pannoramic SCAN (3DHISTECH, Budapest, Hungary) and analysed by Pannoramic Viewer (3D HISTECH, Budapest, Hungary).
9
In situ Hybridization of FENDRR in Oral Cancer
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In situ hybridization (ISH) was performed with digoxigenin-labeled FENDRR probe: 5′-DIG-CAGTGGTTGCTGGGGTTGAAGAGAGGGATGAATA-DIG-3′, following the instructions of the ISH kit purchased from Boster (CA, USA). The slices of 4 pairs of OSCC and normal oral mucosa tissues were treated with conventional dewaxing, rehydration, and then 3% hydrogen peroxide at room temperature for 20 minutes. Then, the slices were digested with pepsin diluted with 3% citric acid at room temperature for 20 minutes to expose RNA fragments. Next, the slices were pre-hybridized for 4 hours and hybridized with FENDRR probe (1.5 ug/ml) overnight. Then the slices were blocked with blocking fluid, followed by incubation with biotinylated mouse anti-digoxigenin and avidin-biotin peroxidase. Finally, a DAB kit (Mxb Bio, China) and hematoxylin were used for visualization. The score used for statistical analyses were calculated as: 1.0*(%Weak positive) + 2.0*(%Positive) + 3.0*(%Strong positive), using Aperio ImageScope (Leica, Germany) (22 (link)).
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In situ hybridization (ISH) was performed with digoxigenin-labeled FENDRR probe: 5′-DIG-CAGTGGTTGCTGGGGTTGAAGAGAGGGATGAATA-DIG-3′, following the instructions of the ISH kit purchased from Boster (CA, USA). The slices of 4 pairs of OSCC and normal oral mucosa tissues were treated with conventional dewaxing, rehydration, and then 3% hydrogen peroxide at room temperature for 20 minutes. Then, the slices were digested with pepsin diluted with 3% citric acid at room temperature for 20 minutes to expose RNA fragments. Next, the slices were pre-hybridized for 4 hours and hybridized with FENDRR probe (1.5 ug/ml) overnight. Then the slices were blocked with blocking fluid, followed by incubation with biotinylated mouse anti-digoxigenin and avidin-biotin peroxidase. Finally, a DAB kit (Mxb Bio, China) and hematoxylin were used for visualization. The score used for statistical analyses were calculated as: 1.0*(%Weak positive) + 2.0*(%Positive) + 3.0*(%Strong positive), using Aperio ImageScope (Leica, Germany) (22 (link)).
10
Quantifying lncRNA PCAT19 Expression
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The expression level of lncRNA PCAT19 of all paraffin‐embedded tissue samples was evaluated with the ISH Kit (BOSTER, catalog number: MK1030). The ISH assays were performed according to the manufacturer's procedure. Briefly, the paraffin‐embedded tissue sections were dewaxed, rehydrated and then digested with protease K followed by rinsing with PBS. Afterwards, the buffer containing the probes was added and incubated at 65°C for 12–16 h. The coverslip was washed with PBS and fixed in 4% paraformaldehyde.
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