Tissues were placed into 10% neutral buffered formalin for 3 days and then stored in 70% ethanol prior to processing. Sections were cut at 4 microns and stained with hematoxylin and eosin or by in situ hybridization (ISH) for ZIKV RNA. The ISH assay was performed using the ViewRNA ISH tissue assay (ThermoFisher) with a probe set targeting the ZIKV (+) strand RNA genome. As a negative control, tissue sections from an uninfected female mouse were stained using the ZIKV probe set. As a positive control, tissues were stained using probes against the housekeeping murine mRNAs GAPDH, PIPB, and β-actin. Tissue sections were de-paraffinized and underwent pre-treatment and protease treatment for ten minutes each. Positive staining (red punctate staining) was detected using an alkaline-phosphatase label probe. The slides were counterstained with Gill’s Hematoxylin I. The IHC assay was performed using a polymer-based indirect immunoalkaline phosphatase detection system with colorimetric detection of antibody/polymer complex with Fast Red chromogen. The primary antibody was a rabbit polyclonal antibody generated against ZIKV VLPs21 (link). Negative controls for IHC comprised both uninfected tissues incubated with the primary antibody, and infected tissues incubated with normal rabbit serum in place of the primary antibody.
Viewrna ish tissue assay
Manufactured by Thermo Fisher Scientific
The ViewRNA ISH tissue assay is a laboratory equipment product designed for in situ hybridization (ISH) analysis of RNA targets in tissue samples. It enables the visualization and localization of specific RNA molecules within the cellular context of tissue sections.
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10 protocols using viewrna ish tissue assay
1
Detection of Zika Virus in Tissues
2
ZIKV RNA Detection in Mouse Tissues
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After mice were euthanized, tissues were immediately placed into 10% neutral buffered formalin for 3 days and then transferred to and stored in a 70% ethanol solution prior to processing. Sections were cut at 4 μm and stained by routine hematoxylin-eosin or by in situ hybridization (ISH). Probe sets targeting the positive-strand ZIKV RNA genome were used with the ViewRNA ISH tissue assay (ThermoFisher). As a negative control, tissue sections from a PBS mock-inoculated C57BL/6 uninfected male mouse were stained using the ZIKV (+) probe set. As a positive control, tissues were stained using probes against the housekeeping murine mRNAs GAPDH, PIPB, and β-actin. Tissue sections were de-paraffinized and underwent an optimized View RNA ISH tissue assay, with pre-treatment and protease treatments lasting 7 min each. Positive staining (red punctate staining) was detected using an alkaline-phosphatase label probe. Nuclei were counterstained with Gill’s Hematoxylin I.
3
In situ Hybridization Detection of ZIKV RNA
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Tissues were placed into 10% neutral buffered formalin for 3 days and then stored in 70% ethanol prior to processing. Sections were cut at 4 microns and stained by in situ hybridization (ISH). The ViewRNA ISH tissue assay (ThermoFisher) with probe sets targeting either positive or negative strand ZIKV RNA or murine PRM2 mRNA (GenBank accession number X07626.1) were utilized. As a negative control, tissue sections from an uninfected male mouse were stained using the ZIKV (+) and (-) probe sets. As a positive control, tissues were stained using probes against the housekeeping murine mRNAs GAPDH, PIPB, and β-actin. Tissue sections were de-paraffinized and underwent an optimized View RNA ISH tissue assay, with pre-treatment and protease treatments lasting ten minutes each. Positive staining (red punctate staining) was detected using an alkaline-phosphatase label probe. Nuclei were counterstained with Gill’s Hematoxylin I.
4
ZIKV RNA Detection in Mouse Tissues
5
Detection of LCMV NP RNA by ISH
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Detection of viral RNA by in situ hybridization was performed on liver sections fixed in 4% PFA using the ViewRNA ISH Tissue Assay (Thermo Fisher Scientific) as previously described (Calabrese and Wieland, 2017 (link)) with the following changes in the protocol: probe-set dilution, 1:30; hybridization time, 2.5 h; preamplification and amplification time, 40 min; and label probe-AP dilution, 1:500. For specific detection of NP coding, LCMV S-segment RNA, we used a custom-made type I probe set (Thermo Fisher Scientific) binding to the negatively oriented LCMV NP gene (GenBank: KY514256.1; nucleotides 1640..3316). Stained slides were acquired in brightfield and in fluorescence (tetramethylrhodamine-isothiocyanate filter) using a slide scanner Hamamatsu NanoZoomer S60. The raw image files were converted in 8-bit grayscale files and overlaid to obtain the merged composite images using ImageJ.
6
In situ hybridization for HBV DNA detection
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The procedures of pretreatment, probeset hybridization and amplification were based on our previous study [16 (link)] and performed using the ViewRNA ISH Tissue Assay (Thermo Fisher, Fremont, CA). The tissue sections were routinely dewaxed and rehydrated, followed with antigen retrieval, protease digestion and refixation with 4%formaldehyde in PBS for five minutes. The HBV DNA probe (VF6–20095) was designed to target the minus strand sequence (nt2959–837) conserved from genotype A to D. After probe hybridization and signal amplification, sections were stained with NBT/BCIP (Roche) in developing solution at 37 °C for 2 h. Sections were then counterstained with Sirius red and washed with water before air-dry and mounting. Rigorous controls were included (positive control slides, no probe ISH control experiments on adjacent slides) were included to ensure the specificity of the ISH assays. The ISH results were examined in comparison with HE staining results in adjacent sections and a score of 0-to-3 was given to tumor and surrounding areas with a standard similar to IHC.
7
In Situ Detection of p73 and FER1L4 in Colorectal Cancer
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Tissue sections (5 μm) were deparaffinized, followed by rehydration in graded alcohols and processed using the ViewRNA ISH Tissue Assay (Invitrogen) procedure. In a nutshell, the sections were subjected to antigen retrieval at 60°C for 60 mins in 1X Pretreatment solution. The tissue sections were then treated with protease digestion and then fixed with 10% NBF. Slides were subsequently incubated with ViewRNA Type 1 Probe set (p73 and FER1L4) block for 2 hours at 40°C. The sections were then treated with pre-amplifier, amplifier and label probe to detect the primary probe. Samples were incubated with Fast Red Substrate for 30 mins at 40°C, followed by counterstaining with hematoxylin. Results were recorded by calculating the percentage of tumor cells portraying distinctive nuclear and cytoplasmic staining. Immunoreactivity for p73 and FER1L4 in non-metastatic and metastatic colorectal cancer tissues was scored as follows: None (<5%, score 0), Weak (+, 5%-25%, score 1), Moderate (++, 25%-75%, score 2), Intense (+++, >75%, score 3).
8
SARS-CoV-2 and MERS-CoV Detection by ISH
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For RNA in situ hybridisation (ISH), tissues were immersion-fixed and embedded in paraffin.
ISH for the detection of SARS-CoV-2 and MERS-CoV was performed using the ViewRNA ISH Tissue Assay (Invitrogen by Thermo Fisher Scientific) following the manufacturer's instructions (MAN0018633 Rev.C.0), as described previously [34 (link)].
ISH for the detection of SARS-CoV-2 and MERS-CoV was performed using the ViewRNA ISH Tissue Assay (Invitrogen by Thermo Fisher Scientific) following the manufacturer's instructions (MAN0018633 Rev.C.0), as described previously [34 (link)].
9
In Situ Hybridization for Zika Virus
10
Optimized RNA-ISH Assay for Collagen Type I
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RNA in situ hybridization (RNA-ISH) assay was performed according to the Invitrogen ViewRNA ISH Tissue Assay protocol (Invitrogen, Carlsbad, CA) with the following optimized pretreatment conditions: heat pretreatment for 10 minutes at 90 to 95°C; protease digestion at 1:100 dilution for 10 minutes at 40°C; COL1A1, a human collagen type I probe (Thermo Fisher; #VA6-18298) was used for the assay. Hybridization for human COL1A1 was performed for 2 hours at 40°C.
Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Thermo Fisher; #VA6-10337) + dihydrodipicolinate reductase (dapB) (Thermo Fisher; #VF1-11712) probes were used for the 2-plex assay control. Hybridization was performed as follows: human COL1A1 for 2 hours at 40°C and human GAPDH + Bacillus subtilis dapB for 2 hours at 40°C.
Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Thermo Fisher; #VA6-10337) + dihydrodipicolinate reductase (dapB) (Thermo Fisher; #VF1-11712) probes were used for the 2-plex assay control. Hybridization was performed as follows: human COL1A1 for 2 hours at 40°C and human GAPDH + Bacillus subtilis dapB for 2 hours at 40°C.
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