PMEC were seeded on 12 mm coverslips (Carl Roth) in 24-well plate (Biochrom) at a density of 10 000 cells/well. After two days of culturing, medium was discarded, and coverslips were washed twice with PBS and fixed with ice-cold methanol (−20 °C, Carl Roth) for 20 min. Cells were permeabilized with 0.2% Triton X-100 (Carl Roth), diluted with PBS for 5 min and washed twice with PBS. Non-specific binding sites were blocked by incubating the coverslips with 10% FBS in PBS for 30 min at room temperature. Coverslips were washed twice with PBS and incubated with mouse anti-cytokeratin 18-fluorescein isothiocyanate (anti-Cy18-FITC, Sigma-Aldrich) and mouse anti-alpha-smooth muscle actin antibodies (clone 1A4, Sigma-Aldrich), respectively in a humidified chamber for 1 h. Coverslips were washed three times with PBS. Bound anti-alpha-smooth muscle actin antibody was visualized by 1 h incubation of the coverslips with goat anti-mouse FITC-labeled secondary antibody (Sigma-Aldrich). Nuclei of the cells were stained with 4’,6-diamidino-2-phenylindole (DAPI, Carl Roth) for 15 min. Coverslips were washed twice with PBS, air dried and mounted with 1,4-diazabicyclo[2.2.2]octane (DABCO) on glass slides (both from Carl Roth). Coverslips were analyzed by immunofluorescence microscopy (Microphot-FXA, Nikon, Düsseldorf, Germany).
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Carl Roth
Sourced in Germany, United States
DAPI is a fluorescent stain that binds to double-stranded DNA. It can be used to visualize and quantify nuclear DNA in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Carl Roth (10 mentions)
Triton x 100, by Merck Group (8 mentions)
Lsm 700, by Zeiss (7 mentions)
Triton x 100, by Carl Roth (7 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Axio observer z1, by Zeiss (6 mentions)
Zen 2009, by Zeiss (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Dmi6000b microscope, by Leica (4 mentions)
Sp5 confocal microscope, by Leica (3 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Draq5, by Thermo Fisher Scientific (3 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (3 mentions)
Plan apochromat 20 0.8 m27 objective, by Zeiss (3 mentions)
Mowiol 4 88, by Carl Roth (3 mentions)
Fluoromount g, by Southern Biotech (3 mentions)
Diagnostic microscope slides, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594 donkey anti goat, by Thermo Fisher Scientific (3 mentions)
128 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Immunocytochemistry of Cultured Cells
2
Immunostaining of ZIKV NS1 and MafG
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The cells were seeded on cover slides and fixed with 4% formaldehyde in PBS for 20 min at room temperature. Permeabilization and blocking were performed as described by Elgner et al. [47 (link)]. The cells were incubated with anti-ZIKV NS1 (1:1000; Biofront Technologies, Tallahassee, FL, USA) and anti-MafG (1.300; Abcam, Cambridge, UK) and afterwards with the secondary antibodies anti-mouse IgG-Alexa 488 (1:1000; Thermo Fisher Scientific, MA, USA) and anti-rabbit-Cy3 (1:400; Jackson Immunoresearch, West Grove, PA, USA). Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (Carl Roth, Karlsruhe, Germany). Finally, the stained coverslips were mounted with Mowiol (Merck, Darmstadt, Germany) and analyzed using the confocal laser scanning microscope LSM 510 Meta and ZEN 2009 software (Carl Zeiss, Oberkochen, Germany).
3
Monocyte Characterization and Viability Assay
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Monocytes were cultivated for 1 DIV with and without SPIO treatment on poly-d-lysine (Sigma-Aldrich) coated glass slides. Then specimens were fixed with 4% PFA (Merck KGaA). Nuclear Fast Red (NFR) (Carl Roth, Karlsruhe, Germany) and Prussian blue staining procedures, embedding with Immu-Mount (Thermo Fisher Scientific) as well as coverslipping were performed as described before (Perl and Good, 1992; Tysiak et al., 2009).
For assessing morphologic characterizations, monocytes were stained after incubation with rat anti-mouse CD11b monoclonal antibody (mAb) overnight at 4°C (1:1000, RRID: AB_2829357; Leinco Technologies, St. Louis, MO, USA), goat anti-rat Alexa Fluor 568 mAb for 2 hours at room temperature (1:500, RRID: AB_2534121; Thermo Fisher Scientific), and 4′,6-diamidino-2-phenylindole (DAPI) (Carl Roth) followed by embedding using Immu-Mount (Thermo Fisher Scientific) and coverslipping.
To analyze the total number as well as the amount of viable and dead cells, monocytes were stained using a cell viability imaging kit (Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the manufacturer’s protocol. Briefly, 50 µL of dye mixture was added to 200 µL of the medium of each specimen followed by 30 minutes of incubation at 37°C in a humidified 5% CO2-enriched atmosphere.
For assessing morphologic characterizations, monocytes were stained after incubation with rat anti-mouse CD11b monoclonal antibody (mAb) overnight at 4°C (1:1000, RRID: AB_2829357; Leinco Technologies, St. Louis, MO, USA), goat anti-rat Alexa Fluor 568 mAb for 2 hours at room temperature (1:500, RRID: AB_2534121; Thermo Fisher Scientific), and 4′,6-diamidino-2-phenylindole (DAPI) (Carl Roth) followed by embedding using Immu-Mount (Thermo Fisher Scientific) and coverslipping.
To analyze the total number as well as the amount of viable and dead cells, monocytes were stained using a cell viability imaging kit (Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the manufacturer’s protocol. Briefly, 50 µL of dye mixture was added to 200 µL of the medium of each specimen followed by 30 minutes of incubation at 37°C in a humidified 5% CO2-enriched atmosphere.
4
Visualizing Subcellular Localization of GFP Fusions
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Subcellular localizations of GFP fusion proteins were examined via fluorescence microscopy as described [69 (link)]. Briefly, approximately 1 × 104 to 1 × 106 spores were inoculated into 300 µL PDM and grown overnight at 25 °C in the light. Either a 100×/1.4 oil objective (Plan-Apochromat) or 63×/0.75 air objective (Plan-Neofluar) were used with the Axio Observer Z1 system (Zeiss) with Laser Lunch System (Model 3iL32, Intelligent Imaging Innovations), QuantEM:512SC camera (Photometrics), and Slide Book 6.0 imaging software (Intelligent Imaging Innovations).
Nuclei were visualized either through ectopically expressed RFP–H2B fusion constructs or staining with 4′,6-diamidino-2-phenylindole (DAPI, Carl Roth GmbH + Co. KG, Karlsruhe, Germany).
Nuclei were visualized either through ectopically expressed RFP–H2B fusion constructs or staining with 4′,6-diamidino-2-phenylindole (DAPI, Carl Roth GmbH + Co. KG, Karlsruhe, Germany).
5
Quantifying Olfactory Neuron Maturity and BoDV-1 Infection
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IF was performed in order to estimate the amount of mature olfactory neurons in the culture of the OM, and to confirm BoDV-1 infection. The ells were fixed with 4% paraformaldehyde (PFA) in a culture medium and permeabilized with 5% nonimmune goat serum (NGS), 3% BSA, and 0.25% (for TUJ1 staining) or 0.1% (for Bo18 staining) Triton® X-100 for 20 min. For the staining of the olfactory neurons, anti- β III tubulin (TUJ1; Covance, Dedham, MA, USA) was used at a dilution 1:1000, in Tris buffered saline (TBS) containing 3% bovine serum albumin (BSA) and 5% nonimmune goat serum (NGS). To detect BoDV-1-N, the same monoclonal antibody against BoDV-1-N (Bo18) was used as described for immunohistochemistry, diluted 1:100 in TBS containing 1% BSA. The cells were incubated with secondary antibodies (Cy2 goat anti-rabbit and Cy3 goat anti-mouse, respectively; Jackson ImmunoResearch, West Grove, PA, USA) for 30 min. Then, 4’,6-diamidino-2-phenylindole (DAPI; Carl Roth, Karlsruhe, Germany) was used to stain the nuclei. Images were taken with Nikon Eclipse 80i fluorescence microscope and NIS Elements BR 3.2 software. The coverslips were mounted with Entellan in Toluen (Merck, Darmstadt, Germany).
6
Multimarker Immunostaining of Brain Sections
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Brain sections were labelled with a mouse anti-RAGE primary antibody (1:100, Santa Cruz Biotechnology) along with proper primary antibodies of different cell markers including rabbit anti-Iba1 (1:500, Wako Pure Chemicals, Osaka, Japan), rabbit anti-NeuN (1:200, Millipore), rabbit anti-GFAP (1:500, Abcam), and rat anti-CD31 (1:300, Dianova, Hamburg, Germany) overnight at 4 °C. These labeled sections were then incubated with Cy3- and AF488-conjugated secondary antibodies (1:1000, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature, counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Carl Roth, Karlsruhe, Germany), and mounted with VECTASHIELD® (Vector Laboratories).
7
Immunofluorescence Imaging of Puromycin-Labeled or SNAP-Tagged Cells
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Following puromycin labeling or SNAP-tag treatment, cells were fixed for 15 min with 4% PFA and washed with 1× PBS. Cells were permeabilized with 0.3% Triton X-100 for 15 min, washed three times in 1× PBS, blocked for 1 hour with 4% bovine serum albumin/10% serum in PBS, and incubated with primary antibody overnight at 4°C. Following washing, cells were treated with secondary antibody for 1 hour in dark at RT. Cells were washed, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in PBS (1 μg/ml; Roth) for 3 min, and mounted with ProLong Gold antifade mountant (Life Technologies, P36930). Samples were imaged soon after the experiment and stored at 4°C. All images were taken for analysis with a Zeiss LSM 880 AiryScan laser scanning confocal microscope at a magnification of ×40. Images were scanned at 1024 × 1024 pixels, with a slice thickness of 1 μM and line and frame average of 4.
8
Visualizing Viral Protein Localization
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QM9 cells, seeded with coverslips in 12 well plates, were infected with rNDV and rNDV_GRABV at an moi of 0.1, fixed with 4% paraformaldehyde 24 h p. i., and permeabilized using 0.1% Triton X-100. After blocking of permeabilized and non-permeabilized cells with 5% bovine serum albumin (BSA) in PBS, they were incubated with α NDV-HN and mAb RABV-G. Binding of primary antibody was visualized using Alexa 488 α-rabbit or 568 α-mouse secondary antibody (Invitrogen). 4’,6-Diamidino-2-phenylindole (DAPI) (Roth, Karlsruhe, Germany) was included in washing steps after binding of secondary antibodies to stain nuclei. Images were taken on a Leica SP5 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) with an oil immersion objective (HCX PL APO 63x/1.40–0.60 objective). Sequential z-sections of stained cells were acquired for maximum projection, and images were processed using ImageJ software [42 (link)].
9
Neuron Visualization and Reconstruction
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After recordings the slice was fixed in 4% PFA in phosphate buffer (PB) at 4°C for 90 min and then stored in phosphate buffered saline (PBS) at 4°C until further processing. It was washed 3 × 15 min to remove excess PFA and incubated with Avidin - Alexa Fluor 488 conjugate (1:1000; Life Technologies, Carlsbad, CA) diluted in PBS containing 5% normal goat serum and 0.2% Triton X at room temperature under light protection for 2 hr. The slice was then washed in PBS for 15 min, incubated with 4,6-diamidino-2-phenylindole (DAPI; 1:10,000; Carl Roth, Germany) in H2O for 3 min, washed again in PBS (15 min) all at room temperature and afterwards embedded in Mowiol 4–88 (Sigma-Aldrich, Taufkichen, Germany).
Fluorescence images were acquired with a Nikon A1+ Confocal Microscope (Nikon, Düsseldorf, Germany) and reconstruction of the neuron was obtained from z-stack confocal images using Neurolucida tracing software (MBF Bioscience, Williston, VT).
Fluorescence images were acquired with a Nikon A1+ Confocal Microscope (Nikon, Düsseldorf, Germany) and reconstruction of the neuron was obtained from z-stack confocal images using Neurolucida tracing software (MBF Bioscience, Williston, VT).
10
Immunofluorescence Analysis of H. pylori Infection
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For immunofluorescence, cells were seeded on poly-L-lysine (Sigma Aldrich, Vienna, Austria) coated cover slips (Carl Roth, Karlsruhe, Germany) and infected with H. pylori P12 wt, N6 wt, N6 ΔhtrA, at a MOI of 50 for 24 h or left untreated (mock). Cells were washed twice with PBS containing CaCl2 and MgCl2 and fixed with ice-cold methanol at −20 °C for 10 min. To localize Cdh1, cells were stained with an antibody targeting the IC domain of E-cadherin (BD Transduction Laboratories™, Heidelberg, Germany), which is highly conserved in all Cdh1 variants. H. pylori was detected using a polyclonal serum raised against H. pylori lysate (gift from Rainer Haas, Munich) and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Carl Roth, Karlsruhe, Germany). After incubation with secondary antibodies coupled to STAR-RED or STAR580 dyes (Abberior, Göttingen, Germany) cover slips were mounted on glass microscopy slides using antifade mounting medium (Abberior, Göttingen, Germany) and fluorescence was detected using a Zeiss Observer Z1 fluorescence microscope equipped with an Abberior Instruments STEDYCON unit for confocal and super-resolution STED microscopy.
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