Synchronous parasites harboring PfBlm-GFP, PfRad51-GFP, or PfMtDNAP were fixed using 4% formaldehyde (Sigma). Membrane permeabilization was performed using 1:3 chilled acetone-methanol, followed by 1-h blocking using 3% bovine serum albumin (BSA). Parasites were probed with mouse anti-cytochrome c (Abcam) and rabbit anti-GFP (Abcam) primary antibodies for 1 h at 37°C followed by three washes with 1× PBST (PBS with Tween 20). Cells were then treated with the secondary antibody cocktail containing Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), Alexa Fluor 594-conjugated rabbit anti-mouse IgG (red), and Hoechst 33342 (blue) (Invitrogen) and subsequently washed thrice with 1× PBST. Finally, parasites were mounted using antifade (Life Technologies). Fluorescence microscope Nikon Eclipse NiE AR was used for analyzing and capturing the green and red fluorescence of GFP and CytC, respectively.
Hoechst 33342 blue
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hoechst 33342 (blue) is a fluorescent dye that binds to DNA. It is commonly used in laboratory applications for staining and visualization of nuclei.
Automatically generated - may contain errors
Lab products found in correlation
C hgfi, by Nikon (2 mentions)
Rabbit anti gfp, by Abcam (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Mouse anti cytochrome c, by Abcam (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
Opera quadruple excitation high sensitivity confocal reader, by PerkinElmer (1 mentions)
Hcs cellmask red, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 protocols using hoechst 33342 blue
1
Immunofluorescence Localization of Parasite Proteins
2
Scaffold-Seeded hMSCs Visualization
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The scaffolds seeded with hMSCs were stained with Hoechst 33342 blue (Invitrogen, USA) and analyzed using fluorescence microscopy (Nikon C-HGFi, Japan) after 20 min of incubation at room temperature. For confocal microscopy (Leica TCS-SP5 II, Leica Microsystem, Mannheim, Germany), the post-fixed (2.5% formalin) scaffold samples were subjected to dual staining using Hoechst dye and acridine orange. The 3D image obtained from the incorporation of multiple series of images collected by confocal laser further assisted in the investigation of cell infiltration up to 0–80 µm into the scaffolds.
3
Quantitative Imaging of Filovirus Infection
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JK, diamond python (Squamata: Pythonidae: Morelia spilota) heart (DpHt), or human epithelial adenocarcinoma HeLa cells infected with EBOV or MARV were stained with murine monoclonal antibodies against EBOV or MARV GP1,2 (6D8 and 9G4 antibody, respectively), followed by Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, Thermo Fisher Scientific Waltham, MA, USA) for high-content quantitative image-based analysis. Infected cells were also stained with Hoechst 33342 (blue) and HCS CellMask Red (Invitrogen, Thermo Fisher Scientific) for nuclei and cytoplasm detection, respectively. Infection rates and cell numbers were determined using high-content quantitative imaging data on an Opera quadruple excitation high sensitivity confocal reader (model 3842 and 5025; PerkinElmer, Waltham, MA, USA) at two exposures using ×10 air, ×20 water, or ×40 water objective lenses as described in (Radoshitzky et al. 2010 (link)). Analysis of the images was accomplished within the Opera environment using standard Acapella scripts.
4
Assessing Cell Viability and Morphology
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About 0.25 ml of Alamar Blue working solution was added to the test samples and control wells, followed by incubation for 4 h and measurement of absorbance at 570 and 600 nm. The scaffolds seeded with bone marrow stromal cells were stained with Hoechst 33342 blue (Invitrogen, USA) and analyzed using a confocal microscope (Nikon C-HGFI, Japan) after 20 min of incubation at room temperature.
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