Cardiac tissues from Hu-NSG mice with or without HIV-1 infection were embedded in paraffin as described earlier. Five micrometer sections were then cut and placed onto glass slides. Slides were de-paraffinized with xylene (3 changes, ten minutes each) and rehydrated in decreasing concentrations of ethanol (100%, 95%, 70% and distilled water, three minutes each) followed by phosphate-buffered saline wash. Immuno-histochemical assay was then used to determine T-lymphocytes (HLA-DR and CD68+ human macrophages in hearts of uninfected and HIV-1 infected Hu-NSG mice. Horse serum (10%) was used as the blocking agents to reduce non-specific interactions. Primary and secondary antibody concentrations were 1:100 to 1:200, respectively. Diaminobenzidine (DAB) was used as the visualizing agent. Images were taken with a Nikon inverted fluorescence microscope (TE 2000). Nikon Elements image analysis software was used to quantitation.
Elements image analysis software
Manufactured by Nikon
Nikon Elements is an image analysis software designed for researchers and scientists. It provides advanced tools for processing, analyzing, and quantifying digital images obtained from microscopes and other imaging devices. The software offers a wide range of features to support various applications, such as cell counting, particle analysis, and measurement of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Inverted fluorescence microscope, by Nikon (1 mentions)
C2si camera, by Nikon (1 mentions)
C2 microscope, by Nikon (1 mentions)
Zero blunt topo pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Olympus (1 mentions)
Fluoview 1000, by Olympus (1 mentions)
5 protocols using elements image analysis software
1
Quantifying Immune Cell Infiltration in HIV-Infected Cardiac Tissues
2
Quantitative Analysis of Neuroinflammation
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Confocal images were obtained on a Nikon C2+ microscope equipped with CFI Plan Apochromat Lambda Series Objectives. Images were captured with Nikon C2si+ camera and analyzed using Nikon Elements software. In Thy1-GFP(M) mice, layer I of the medial PFC was identified and apical dendritic segments were imaged using 40x objective lens with a 2.5x zoom (NA 1.4) with z-stack sampling: 0.075 μm. For each sample, 6–8 dendritic segments were analyzed in NeuronStudio as previously described (Wohleb et al., 2018 (link)). Proportional area of GFAP+ or IBA-1+ material in images taken with the 20x objective was measured using standardized threshold parameters with ImageJ software (NIH, Bethesda, Maryland). IBA-1+ cells were counted by a trained researcher blinded to animal condition (4–6 images per sample). To quantify the number and volume of GFP + inclusions within IBA-1+ microglia (16–25 per sample) or GFAP + astrocytes, confocal images (40x; zoom 2.5x), NA 0.95, z-stack sampling: 0.2 μm) were examined in 3-dimensional space and orthogonal z-stacks with Nikon Elements Image Analysis software. Confocal imaging with these settings provides sufficient resolution to detect synaptic inclusions (average inclusion diameter: 0.25 μm) (Weinhard et al., 2018 (link)).
3
Cloning and Visualization of Developmental Genes
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cDNAs for foxc1a, notch1a, and dla were amplified via high fidelity Phusion-HF PCR (NEB) from 48 hpf AB WT cDNA libraries using primers in the Key resources table. PCR products were cloned using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen), as per manufacturer protocols, and sequenced validated. Plasmids encoding phox2bb, sox10, mmp2 were generously sourced as listed in the key resources table. Antisense digoxigenin (DIG)-labeled riboprobes were produced from cDNA templates of each gene. AB wild type embryos were treated and stained to visualize expression as previously described in Jowett and Lettice, 1994 (link). Following in situ reactions, embryos were post-fixed in 4% Paraformaldehyde (PFA) and mounted in 75% Glycerol for imaging. A Nikon Ni-Eclipse Motorized Fluorescent upright compound microscope with a 4X objective was used in combination with a DS-Fi3 color camera. Images were exported via Nikon Elements Image Analysis software.
4
Mitochondrial superoxide assessment by MitoSOX
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Mitochondrial superoxide generation was assessed by MitoSOX Red (Molecular Probes) staining, a fluorogenic dye that is taken up by mitochondria, where it is readily oxidized by superoxide. Fresh frozen 5‐μm LV slices were incubated for 20 minutes at 37°C with1.5 μM MitoSOX Red and fluorescence imaged at 60× magnification via an Olympus FluoView 1000 laser scanning confocal microscope (Olympus America). Total mean fluorescence (red) intensity (MFI) in five random fields (for each experiment) was measured with Nikon Elements image analysis software (Nikon Instruments). Fluorescence intensity unit values from LV for each experimental group were averaged and presented as a measure for O2− production.
5
Quantitative Kidney Histopathology Analysis
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