Lung tissues were immediately fixed after harvesting, embedded in paraffin, subjected to sectioning (5 μm sections), and stained with hematoxylin and eosin (H&E) as well as with antibodies against SARS-CoV-2 spike protein and CD11b (pan myeloid cells), respectively. For H&E staining, lung sections were processed following standard histological procedure. For indirect immunohistochemistry (IHC), lung sections were also processed following standard immuno-histochemical technique with antigen retrieval (i.e., 15 min heat-induced with EDTA pH 6.0). Primary antibodies were used as follows: anti-spike protein at 1:200 dilution and anti-CD11b at 1:200 dilution. Images taken were scanned with the Aperio AT2 system (Leica, Buffalo Grove, IL). The areas with detected spike protein and CD11b were divided by the sum of the areas corresponding to cellular structures counterstained with hematoxylin + anti-spike protein and anti-CD11b, respectively. Calculated ratios are represented as a percentage of control. All histology and IHC was performed - by a third party at the Inotiv facility (Boulder, CO, USA).
At2 system
Manufactured by Leica
The AT2 system is a laser-based microscopy platform designed for high-resolution imaging and analysis. It provides advanced optical capabilities for applications in materials science, life sciences, and other research areas. The core function of the AT2 system is to enable precise, non-invasive imaging and characterization of samples at the micro- and nanoscale.
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Lab products found in correlation
6 protocols using at2 system
1
Lung tissue histopathology and SARS-CoV-2 spike protein analysis
2
Histology Sample Preparation and Imaging
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Selected tissue blocks were processed by 2 commercial, CAP/CLIA certified histology laboratories. Sections were cut from each block and processed using standard H&E and IHC staining protocols. IHC stains were performed with pan-cytokeratin antibody (AE1/AE3). Before scanning, slides were manually inspected for defects such as air bubbles, and any substandard slides were rejected. Slides that passed the quality check were scanned with a Leica AT2 system at a resolution of 0.25 µm/pixel. Resulting whole-slide images (WSI’s) were manually reviewed for overall image quality. During this quality control process, images containing defects such as missing tissue or large out of focus regions were rescanned.
3
Quantifying Sternal Bone Marrow Adipocytes
4
Quantifying Sternal Bone Marrow Adipocytes
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Juvenile sterna were fixed in 4% PFA for 24 h at 4°C, decalcified in 0.5 M EDTA for 4–7 days, and embedded in paraffin. Adjacent 5-mm paraffin sections were stained with hematoxylin and eosin to evaluate adipocyte morphology. Whole slide scan images were captured using the Leica AT2 System (Leica Biosystems, Deer Park, IL). Bone marrow adipocytes were outlined manually and the mean cross-sectional adipocyte area and the adipocyte number per sternal bone marrow area were calculated using ImageJ software.
5
Multistaining Quantifies CD4+ T Cells
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Immunohistochemical staining and QIA were performed as previously described [16] . In brief, immunohistochemistry was performed using a biotin-free polymer approach (Golden Bridge International) on 5μm tissue sections mounted on glass slides, which were dewaxed and rehydrated with double-distilled water. Multistaining of CD4/CD68/CD163 to quantify CD4 + T cells was performed. This multistaining approach allows the intense staining of the macrophage/myeloid cell markers to mask the faint CD4 expressed on these cells and to distinctly identify CD4 + T cells from myeloid lineage cells. Heat-induced epitope retrieval was performed by heating sections in 0.01% citraconic anhydride containing 0.05% Tween-20, then incubated with primary antibody to CD4 (Goat anti-CD4, R&D system ref: AF-379-NA), CD68 (Rabbit anti-CD68, Sigma ref: HPA048982) and CD163 (rabbit anti-CD163, Lifespan Biosciences ref: LS-B2661). All slides were scanned at high magnification (×200) using the AT2 System (Aperio Technologies), yielding high-resolution data from the entire tissue section. Representative regions of interest (500 × 500 μm) were identified, and high-resolution images were extracted from these whole-tissue scans. The percentage area of the positive cell zone was quantified using CellProfiler version 3.1.5.
6
Quantification of Immune Cell Subsets
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