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.
Eclipse microscope
Manufactured by Olympus
The Eclipse microscope is an optical microscope designed for laboratory use. It features high-quality lenses and optics to provide clear and detailed images of specimens. The Eclipse microscope is capable of various magnification levels to accommodate different observation needs.
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Lab products found in correlation
Rnascope 2.5 hd assay brown kit, by Advanced Cell Diagnostics (3 mentions)
Dm6b microscope, by Leica (2 mentions)
Dfc7000t, by Leica (1 mentions)
Donkey anti rabbit igg h l dylight 488, by Abcam (1 mentions)
Ds fi1 camera, by Nikon (1 mentions)
Dfc7000 t camera, by Leica (1 mentions)
Neutral buffered formalin, by Thermo Fisher Scientific (1 mentions)
Ab56416, by Abcam (1 mentions)
Bmk 2202, by Vector Laboratories (1 mentions)
Vectastain universal kit, by Vector Laboratories (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
Dp71 color camera, by Olympus (1 mentions)
Elements software, by Nikon (1 mentions)
11 protocols using eclipse microscope
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
Osteogenic Differentiation Analysis
4
Histological Analysis of MCL Healing
5
Golgi Staining for Dendritic Spine Quantification
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Golgi staining was conducted following the prescribed steps outlined by the manufacturer (FD Rapid GolgiStain™ Kit, USA). Brain tissue samples were submerged in the impregnation solution provided in the Rapid GolgiStain kit. The samples were sectioned into 100 µm slices and placed on glass slides for microscopic examination as per the manufacturer's directions. The prepared slices underwent a dehydration process via a series of ethanol baths at concentrations of 50%, 75%, 95%, and 100% for 4 minutes at each concentration. Postdehydration, the tissue samples were clarified in xylene before image acquisition using an Olympus Eclipse microscope.
Synapse densities were quantified utilizing the Golgi staining method. The total count of dendritic spines on a specified segment of dendrites was performed under 100x magnification. The resulting value was then divided by the length of the dendrite segment to derive the dendritic spine density. The investigator conducting the counting was blinded to the treatment conditions of the mice.
Synapse densities were quantified utilizing the Golgi staining method. The total count of dendritic spines on a specified segment of dendrites was performed under 100x magnification. The resulting value was then divided by the length of the dendrite segment to derive the dendritic spine density. The investigator conducting the counting was blinded to the treatment conditions of the mice.
6
SARS-CoV-2 RNA and ACE2 Expression in Lung
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Animals were euthanized, and tissues were harvested before lung inflation and fixation. The left lung was tied off at the left main bronchus and collected for viral RNA analysis. The right lung was inflated with ∼1.2 mL of 10% neutral buffered formalin using a 3-mL syringe and catheter inserted into the trachea. For fixation after infection, inflated lungs were kept in a 40-mL suspension of neutral buffered formalin for 7 days before further processing. Tissues were embedded in paraffin, and sections were 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, treated with H2O2 and Protease Plus prior to probe hybridization. Probes specifically targeting hACE2 (cat no. 848151) or SARS-CoV-2 S sequence (cat no 848561) were hybridized followed by proprietary signal amplification and detection with 3,3′-Diaminobenzidine. Tissues were counterstained with Gill’s hematoxylin. An uninfected mouse was used as a negative control and stained in parallel. 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.
7
Placental Histology and Immunohistochemistry
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Placentas and fetuses were dissected by cesarean section on E14.5. Harvested samples were fixed in 10% neutral buffered formalin (Thermo Fisher Scientific) at room temperature and embedded in paraffin. At least five placentas from different dams with the indicated genotypes or treatments were sectioned and stained with hematoxylin and eosin to assess morphology. Histologic images were captured by use of a Nikon Eclipse microscope equipped with an Olympus DP71 color camera under 2×, 20×, and 40× objectives. Measurement of size and thickness of different placental layers were performed using ImageJ (National Institutes of Health).
For immunohistochemical staining of mouse placentas, deparaffinized tissues were quenched with 3% H2O2, blocked for 2 h, and incubated with primary antibodies anti-p62 (1:500; ab56416; Abcam) overnight. The M.O.M. kit (BMK-2202; Vector Laboratories) was applied for blocking and secondary antibody incubation according to the manufacturer’s instructions. The sections were then incubated with the ABC reagent from the Vectastain universal kit (PK-7200; Vector Laboratories) and developed using the DAB substrate kit (SK-4100; Vector Laboratories). Tissues were counterstained with hematoxylin. A no–primary antibody staining was included as a negative control.
For immunohistochemical staining of mouse placentas, deparaffinized tissues were quenched with 3% H2O2, blocked for 2 h, and incubated with primary antibodies anti-p62 (1:500; ab56416; Abcam) overnight. The M.O.M. kit (BMK-2202; Vector Laboratories) was applied for blocking and secondary antibody incubation according to the manufacturer’s instructions. The sections were then incubated with the ABC reagent from the Vectastain universal kit (PK-7200; Vector Laboratories) and developed using the DAB substrate kit (SK-4100; Vector Laboratories). Tissues were counterstained with hematoxylin. A no–primary antibody staining was included as a negative control.
8
Chikungunya Virus RNA Detection in Mouse Spleen
9
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.
10
Lung Tissue Morphometric Analysis
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Tissue sections were stained with hematoxylin and eosin and analyzed blindly. Images were obtained on a Nikon Eclipse microscope or Olympus DP-71, and morphometric analysis was performed using Nikon Elements software. Analysis was performed on 10 different areas of the lungs, and at least 10 high-powered fields were analyzed in each area.
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