Four week-old WT and Mxra8Δ8/Δ8 male mice were inoculated subcutaneously in the foot with 103 FFU of CHIKV AF15561. At 3 dpi, animals were euthanized and perfused extensively with PBS. Ipsilateral and contralateral feet were harvested and fixed in 4% paraformaldehyde (PFA) for 24 h. Tissues were decalcified in 14% EDTA free acid for two weeks and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin to assess tissue morphology. To determine sites of CHIKV infection, RNA in situ hybridization was performed using the RNAscope 2.5 HD Assay (Brown Kit) according to the manufacturer’s instructions (Advanced Cell Diagnostics). Briefly, sections were deparaffinized, treated with H2O2 and Protease Plus prior to probe hybridization. A probe specifically targeting the CHIKV RNA (strain AF15561) (Advanced Cell Diagnostics, #481891) was custom-designed and used for ISH experiments. Tissues were counterstained with Gill’s hematoxylin. To assess the cell types that express Mxra8 RNA in naive mice, in situ hybridization was performed using the RNAscope 2.5 HD Assay (Red Kit). A probe specifically targeting Mxra8 (NM_024263.4) (Advanced Cell Diagnostics, #520711) was custom-designed. A negative control probe (#320751) was used in parallel. Tissue sections were visualized using a Nikon Eclipse microscope equipped with an Olympus DP71 color camera.
Rnascope 2.5 hd assay brown kit
Manufactured by Advanced Cell Diagnostics
The RNAscope 2.5 HD Assay (Brown Kit) is a highly sensitive and specific in-situ hybridization technology developed by Advanced Cell Diagnostics. The core function of this kit is to detect and visualize single-molecule RNA targets in formalin-fixed, paraffin-embedded (FFPE) tissue samples.
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5 protocols using rnascope 2.5 hd assay brown kit
1
Chikungunya Virus Infection in Mice
2
SARS-CoV-2 Lung Histopathology Analysis
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Mice were euthanized, and tissues were harvested prior to lung inflation and fixation. The right lung was inflated with approximately 1.2 mL of 10% neutral buffered formalin using a 3-mL syringe and catheter inserted into the trachea. To ensure fixation of virus, inflated lungs were kept in a 40-mL suspension of neutral buffered formalin for 7 days before further processing. Tissues were paraffin-embedded and sections were subsequently stained with hematoxylin and eosin. RNA in situ hybridization was performed using the RNAscope 2.5 HD Assay (Brown Kit) according to the manufacturer’s instructions (Advanced Cell Diagnostics). Briefly, sections were deparaffinized and treated with H2O2 and Protease Plus prior to RNA probe hybridization. Probes specifically targeting SARS-CoV-2 S sequence (cat no 848561) were hybridized followed by signal amplification and detection with 3,3′-Diaminobenzidine. Tissues were counterstained with Gill’s hematoxylin and an uninfected mouse was stained in parallel and used as a negative control. The lung pathology was evaluated, and representative photomicrographs were taken of stained slides under investigator-blinded conditions. Tissue sections were visualized using a Nikon Eclipse microscope equipped with an Olympus DP71 color camera or a Leica DM6B microscope equipped with a Leica DFC7000T camera.
3
MmuPV1 Detection using RNAscope Assay
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MmuPV1 detection was performed using the RNAscope 2.5 HD Assay-Brown kit (Advanced Cell Diagnostics, Newark, CA; 322300) and probe to MmuPV1 E4 (473281) as previously described [14 (link)]. NSG mouse anal tissues that were infected with MmuPV1 or mock infected [14 (link)] were included as positive and negative controls, respectively.
4
Chikungunya Virus Infection in Mice
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Four week-old WT and Mxra8Δ8/Δ8 male mice were inoculated subcutaneously in the foot with 103 FFU of CHIKV AF15561. At 3 dpi, animals were euthanized and perfused extensively with PBS. Ipsilateral and contralateral feet were harvested and fixed in 4% paraformaldehyde (PFA) for 24 h. Tissues were decalcified in 14% EDTA free acid for two weeks and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin to assess tissue morphology. To determine sites of CHIKV infection, RNA in situ hybridization was performed using the RNAscope 2.5 HD Assay (Brown Kit) according to the manufacturer’s instructions (Advanced Cell Diagnostics). Briefly, sections were deparaffinized, treated with H2O2 and Protease Plus prior to probe hybridization. A probe specifically targeting the CHIKV RNA (strain AF15561) (Advanced Cell Diagnostics, #481891) was custom-designed and used for ISH experiments. Tissues were counterstained with Gill’s hematoxylin. To assess the cell types that express Mxra8 RNA in naive mice, in situ hybridization was performed using the RNAscope 2.5 HD Assay (Red Kit). A probe specifically targeting Mxra8 (NM_024263.4) (Advanced Cell Diagnostics, #520711) was custom-designed. A negative control probe (#320751) was used in parallel. Tissue sections were visualized using a Nikon Eclipse microscope equipped with an Olympus DP71 color camera.
5
MmuPV1 Viral Signal Detection Using RNAScope
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Detection of MmuPV1 viral signal was performed using the RNAScope 2.5 HD assay-brown kit (322300; Advanced Cell Diagnostics, Newark, CA) according to the manufacturer's protocol using a probe to MmuPV1 E4 (473281) or MmuPV1 E6/E7 (409771). As controls, select tissues were treated with 20 U of DNase I (EN0521; Thermo Fisher), 500 μg RNase A (1006657; Qiagen), and 2,000 U RNase T1 (EN0542; Thermo Fisher) for 30 min at 40°C prior to probing.
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