In situ hybridization (ISH) was performed using locked nucleic acid (LNA) probes for U6 snRNA 47 (link) and SARS-CoV-2 RNA, designed to target the sense strand of ORF1ab and Spike regions of the viral genome. Scrambled sequences were used as control. Experiments were performed using a dedicated ISH kit for Formalin-fixed paraffin-embedded (FFPE) tissues (Qiagen) according to the manufacturer’s protocol. Briefly, FFPE tissue slides were deparaffinized in xylene, treated with proteinase-K (15 μg/ml) for 5 min at 37°C and incubated with either SARS-CoV-2 (40 nM) or U6 probes (2 nM) for one hour at 54°C in a hybridizer. After washing with SSC buffer, the presence of SARS-CoV-2 RNA was detected using an anti-DIG alkaline phosphatase (AP) antibody (1:500) (Roche Diagnostics) supplemented with sheep serum (Jackson Immunoresearch) and bovine serum albumin (BSA). Hybridization was detected by adding NBT-BCIP substrate (Roche Diagnostics). Nuclei were counterstained with nuclear fast red.
Sheep serum
Manufactured by Jackson ImmunoResearch
Sourced in United States
Sheep serum is a biological fluid collected from the blood of sheep. It contains a variety of proteins, including immunoglobulins, albumin, and other components found in the circulatory system of sheep.
Automatically generated - may contain errors
Lab products found in correlation
Nbt bcip substrate, by Roche (2 mentions)
Anti dig alkaline phosphatase antibody, by Roche (2 mentions)
A1si laser scanning confocal microscope, by Nikon (1 mentions)
Alexa 647 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti acetylated tubulin, by Merck Group (1 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
Pnfκb p65 s536, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Mien1, by Abnova (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Nuclear fast red, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Icc50w light microscope, by Leica (1 mentions)
Mircury lna detection probe, by Qiagen (1 mentions)
4 protocols using sheep serum
1
In Situ Hybridization Detection of SARS-CoV-2 RNA
2
Whole-Mount Immunohistochemistry for Axon Labeling
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For whole-mount immunohistochemistry, larvae were immersed overnight in 4% formaldehyde at 4 °C, washed three times at room temperature in phosphate-buffered saline (PBS)/0.02% Triton X-100, pH 7.3, incubated in 2% BSA with 5% sheep serum (Cat #013–000-12, Jackson Immuno, West Grove, PA, USA) for 2 h at room temperature, and then incubated overnight with primary antibodies at 4 °C. For labeling axons, mouse anti-acetylated tubulin (1:1000, Cat #T6793, Sigma) and Alexa 647-conjugated secondary antibodies (1:500, Cat #A-21235, Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) were used.
All fluorescent images were taken in the 2-somite area above the end of the yolk extension using an A1Si laser-scanning confocal microscope (Nikon, Tokyo, Japan). Confocal images (1-μm z-stacking) were processed using NIS-Elements AR Analysis 4.30 software (Nikon).
All fluorescent images were taken in the 2-somite area above the end of the yolk extension using an A1Si laser-scanning confocal microscope (Nikon, Tokyo, Japan). Confocal images (1-μm z-stacking) were processed using NIS-Elements AR Analysis 4.30 software (Nikon).
3
Antibody Validation for Protein Analysis
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The following antibodies and reagents were used: Mouse monoclonal and mouse polyclonal MIEN1 (Abnova; antibody specificity tested and proven in previous studies[15 (link), 17 (link)]), rabbit polyclonal MIEN1 (Life Technologies; antibody specificity tested in previous studies[15 (link)]), mouse monoclonal GAPDH (Santa Cruz Biotechnology), rabbit monoclonal pNF-κB p65 S536 and rabbit polyclonal MMP-9 (Cell Signaling Technology), mouse monoclonal VEGF and uPA (R&D Systems), mouse monoclonal Alexa Fluor 594 conjugated Phalloidin (Life Technologies), mouse monoclonal E-cadherin (BD Biosciences), Vimentin (supernatant developed in mouse and tested against human antigen, Developmental Studies Hybridoma Bank), anti-mouse and anti-rabbit IgG (Promega), AlexaFluor 488 goat anti-mouse IgG and AlexaFluor 594 goat anti-mouse IgG (Life Technologies) sheep anti-DIG-AP antibody and NBT-BCIP ready-to-use tablets (Roche), sheep serum (Jackson ImmunoResearch), rabbit IgG, BSA, levamisole hydrochloride, Tris-HCl (pH 7.4), nuclease free water, SSC buffer, Xylene, Tween-20, Nuclear Fast Red, Hematoxylin and Eosin (Sigma-Aldrich) and Permount and PBS (Thermo Fisher Scientific).
4
In situ Detection of SARS-CoV-2 RNA
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In situ hybridization was performed using locked nucleic acid (LNA) probes for U6 snRNA (miRCURY LNA Detection probe, Qiagen, Cat. No. YD00699002) and SARS-CoV-2 RNA, designed to target the sense strand of ORF1ab and Spike regions of the viral genome. A scrambled sequence probe (YD00699004) was used as a control.
Experiments were performed using a dedicated ISH (Qiagen) according to the manufacturer’s protocol. Briefly, air-dried frozen tissue sections were incubated with either SARS-CoV-2 (40 nM) or U6 probes (2 nM) for 1 h at 54°C in a hybridizer. After washing with SSC buffer, the presence of SARS-CoV-2 RNA was detected using an anti-DIG alkaline phosphatase (AP) antibody (1:500) (Roche Diagnostics) supplemented with sheep serum (Jackson Immunoresearch) and bovine serum albumin (BSA). Hybridization was detected by adding NBT-BCIP substrate (Roche Diagnostics). Nuclei were counterstained with nuclear fast red. Images were acquired using a Leica ICC50W light microscope.
+ Expand
In situ hybridization was performed using locked nucleic acid (LNA) probes for U6 snRNA (miRCURY LNA Detection probe, Qiagen, Cat. No. YD00699002) and SARS-CoV-2 RNA, designed to target the sense strand of ORF1ab and Spike regions of the viral genome. A scrambled sequence probe (YD00699004) was used as a control.
Experiments were performed using a dedicated ISH (Qiagen) according to the manufacturer’s protocol. Briefly, air-dried frozen tissue sections were incubated with either SARS-CoV-2 (40 nM) or U6 probes (2 nM) for 1 h at 54°C in a hybridizer. After washing with SSC buffer, the presence of SARS-CoV-2 RNA was detected using an anti-DIG alkaline phosphatase (AP) antibody (1:500) (Roche Diagnostics) supplemented with sheep serum (Jackson Immunoresearch) and bovine serum albumin (BSA). Hybridization was detected by adding NBT-BCIP substrate (Roche Diagnostics). Nuclei were counterstained with nuclear fast red. Images were acquired using a Leica ICC50W light microscope.
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