Samples of kidney tissue were collected from mice, embedded in Tissue-Tek® O.C.T.™, immediately frozen in 2-methylbutane (isopentane) resting in liquid nitrogen and stored at −70°C. Sections were cut to a thickness of 10–15 μm, fixed with 4% paraformaldehyde and stained with Hoechst 33342 (1 μg/mL, Molecular Probes), anti-MPO (1:50; sc-16128-R, Santa Cruz Biotechnology) and anti-DNA/histone H1 (1:200, MAB3864, Merck Millipore) antibodies, followed by anti-rabbit Alexa Fluor 594 (1:100; Molecular Probes) or anti-mouse Alexa Fluor 488 (1:400; Molecular Probes). Confocal images were taken with a Leica TCS SP5-AOBS microscope (Leica Microsystems, Mannheim, Germany); epifluorescence images were taken with a Zeiss Axioplan. Permeabilizing agents were not used, resulting in exclusive extracellular labeling. Assessment was made in a purely qualitative fashion to detect the presence or absence of NETs in the analyzed samples.
Tcs sp5 aobs microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS SP5-AOBS is a confocal laser scanning microscope that offers advanced imaging capabilities. It features an Acousto-Optical Beam Splitter (AOBS) for flexible control of excitation wavelengths. The microscope is designed to provide high-resolution, multi-channel imaging for a wide range of applications.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Sc 16128 r, by Santa Cruz Biotechnology (2 mentions)
Axioplan, by Zeiss (2 mentions)
Ab5103, by Abcam (2 mentions)
P36935, by Thermo Fisher Scientific (2 mentions)
Poly d lysine, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Dmi6000b, by Leica (1 mentions)
Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dm5000b microscope, by Leica (1 mentions)
Sc 74495, by Santa Cruz Biotechnology (1 mentions)
Ap124f, by Merck Group (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Vibratome, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
11 protocols using tcs sp5 aobs microscope
1
Extracellular Traps in Kidney Tissue
2
Immunofluorescence Analysis of Neutrophil Extracellular Traps
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The tissue segment was embedded in optimum cutting temperature, and sections (15 μm) were blocked and incubated with primary antibodies against Histone H3 citrulline R17+R2+R8 (1:1000; ab5103, Abcam) and then stained with conjugated secondary antibodies. Images and analysis were performed using a fluorescence microscope (Leica DMI6000B). For immunostaining, neutrophils were attached on slides coated with poly-d -lysine (Sigma) and stimulated with PMA (100 ng/ml) or medium. The slides were then fixed with 4% paraformaldehyde and stained with DAPI (P36935, Molecular Probes), anti-MPO (1:50; sc-16128-R, Santa Cruz Biotechnology), and anti-histone H4 (1:200, sc-25260, Santa Cruz Biotechnology) antibodies, followed by anti-rabbit Alexa Fluor 594 (1:100; Molecular Probes) or anti-mouse Alexa Fluor 488 (1:400; Molecular Probes). Confocal images were taken with a Leica TCS SP5-AOBS microscope (Leica Microsystems, Mannheim, Germany). Epifluorescence images were taken with a Zeiss Axioplan. Permeabilizing agents were not used for exclusive extracellular labeling. The percentage of NETs was determined from six non-overlapping fields per well and the average was from triplicates for each condition in every experiment. All analysis was performed blinded to treatment conditions.
3
Neutrophil Chikungunya Virus Interaction
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A total of 5 × 104 isolated neutrophils were attached on a slide coated with poly-D-lysine (Sigma-Aldrich) and incubated with CHIKV. After 4 h of incubation, the slides were washed with phosphate-buffered solution (PBS) and fixed with 4% paraformaldehyde for 30 min. The samples were blocked with a PBS/BSA 2% solution (Sigma-Aldrich) for 2 h at room temperature and incubated with primary antibodies against histone H3 citrulline R17+R2+R8 (ab5103, Abcam, 1:500), murine polyclonal anti-CHIKV antibodies (obtained from Dr. Figueiredo's lab, 1:100), and anti-Ly6G antibodies (16-9668-82, Invitrogen, 1:50) overnight at 4°C. After washing with PBS, anti-rabbit Alexa Fluor 488 (1:1000, Molecular Probes), anti-rat Alexa Fluor 594 (1:100, Molecular Probes), and/or anti-mouse Alexa Fluor 594 (1:200, Molecular Probes) were incubated for 2 h at room temperature. The slides were counterstained with DAPI (P36935, Molecular Probes), and the images were acquired with a Leica TCS SP5-AOBS microscope (Leica Microsystems, Mannheim, Germany).
4
Quantifying Macrophage Subsets in Implant Sites
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To analyze Mφ1 and Mφ2 macrophages, skin sections were stained for F4/80, CD274, and CD206 to quantify the macrophage phenotype surrounding the implant site. Immunolabeling was performed with antibodies against F4/80 (1:50 dilution; Abcam, Cambridge, UK), combined either with CD274 (Mφ1) (1:100 dilution; Novus Biologicals, Littleton, CO, USA) or CD206 (Mφ2) (1:200 dilution; Abcam), followed by detection with Alexa-488-conjugated secondary antibodies (1:200 dilution; Invitrogen). Quantification was performed in mice sacrificed 15 days post-treatment. A total of four serial skin sections were prepared, and four images of the zone of the implant were taken per section with a 20× objective. Immunofluorescent images were acquired with a Leica TCS-SP5-AOBS microscope (Leica Microsystems, Wetzlar, Germany), the immunopositive cells were quantified, and the ratio of Mφ1/Mφ2 macrophages was calculated in each field.
5
Quantification of Proliferative Olig2+ Cells in Midbrain Regions
6
Cellular Uptake of DOX Nanoparticles
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Cells were grown on glass coverslips for 24 h. Then, A2780res were incubated for 1 h with DOX-NPs, GC DOX-NPs, and DOX solution. After rinse, cells were fixed in 4% formaldehyde for 10 min at room temperature and DAPI was used to counterstain the nuclei. Fluorescent images were acquired using a Leica TCS SP5 AOBS microscope (Leica Microsystems).
7
In vivo Fluorescence Imaging of Worm Proteins
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Ensemble GFP, dCALM and CALM-spFRET imaging of live worms were performed on a confocal Leica TCS SP5AOBS microscope or a Leica DM5000B microscope equipped with a CSU10 spinning disc confocal scanner system (Yokogawa). Appropriate laser excitation wavelengths and emission filtering were used to image GFP and activated split-GFP fusions (488 nm excitation), anti-GFP antibodies or UNC-29-tagRFP (561 nm excitation) and M3-A647 peptides (633 nm excitation). FRET from complemented CD4-split-GFP to M3-A647 was detected by single laser excitation at 488 nm and simultaneous detection in the GFP and the A647 emission channels. Live worms were anaesthetized with 0.2% sodium azide or 50 mM muscimol before being mounted between 2% agarose pads and microscope coverslips for imaging. Freeze-fractured and immunostained worms were imaged on the same microscopes, except for immunostained CD4-split-GFP worms that were imaged by wide-field epifluorescence.
8
Immunofluorescence Analysis of Osteogenic Markers
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Immunofluorescence analyses were performed as previously reported [30 (link)]. Briefly, cells were fixed and processed according to the manufacturer’s protocols. Primary antibodies for RUNX2 and osteocalcin (sc74495, Santa Cruz, Dallas, Texas, USA) were diluted according to the manufacturer’s instruction in antibody dilution buffer and incubated overnight at 4°C. Slides were then incubated with the secondary antibodies goat mouse fluorescein conjugated (cat. Ap124f, Millipore, Burlington, Massachusetts, USA). Nuclear staining was performed by ProLong™ Gold Antifade Mountant with DAPI. The staining was analyzed using a Leica (Wetzlar, Germany) TCS SP5 AOBS microscope. To express data in a semiquantitative way, six different fields were analyzed for each sample, in three independent experiments with about 80–100 total cells.
9
Tau4RD Phase Separation Modulation
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Tau4RD was mixed with a small amount
(1.2% of the total protein) of tau4RD labeled with fluorescein
isothiocyanate (FITC) or with Alexa Fluor 488 (Thermo Fisher Scientific)
to report its liquid–liquid phase separation, as previously
reported.16 (link) Liquid–liquid phase
separation of tau4RD was induced using heparin, in the
absence or presence of espresso coffee, caffeine, or genistein at
different concentrations (35–280 μg/mL). Where indicated,
coffee, caffeine, and genistein were added to already-formed droplets
(after 5 min). In all samples, 35 μM protein in 20 mM sodium
phosphate buffer, pH 6.0, 30 mM NaCl, and 5 mM DTT was mixed with
8.75 μM heparin. For phase separation imaging, 7 μL of
the solution was spotted onto a microscope slide, covered with a circular
coverslip, and sealed with nail polish. Condensate images were acquired
on a Leica TCS SP5 AOBS microscope to visualize the formation of droplets
over time. Image analysis was performed with FIJI ImageJ software
(v2.0).
(1.2% of the total protein) of tau4RD labeled with fluorescein
isothiocyanate (FITC) or with Alexa Fluor 488 (Thermo Fisher Scientific)
to report its liquid–liquid phase separation, as previously
reported.16 (link) Liquid–liquid phase
separation of tau4RD was induced using heparin, in the
absence or presence of espresso coffee, caffeine, or genistein at
different concentrations (35–280 μg/mL). Where indicated,
coffee, caffeine, and genistein were added to already-formed droplets
(after 5 min). In all samples, 35 μM protein in 20 mM sodium
phosphate buffer, pH 6.0, 30 mM NaCl, and 5 mM DTT was mixed with
8.75 μM heparin. For phase separation imaging, 7 μL of
the solution was spotted onto a microscope slide, covered with a circular
coverslip, and sealed with nail polish. Condensate images were acquired
on a Leica TCS SP5 AOBS microscope to visualize the formation of droplets
over time. Image analysis was performed with FIJI ImageJ software
(v2.0).
10
Immunostaining of Pancreas and Brain Tissue
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For immunostaining, pancreas or brain were fixed overnight in 4% paraformaldehyde at 4°C. Pancreas were dehydrated, paraffin embedded, and sectioned at 5 μm thickness. Brains were coronally sectioned into six series of 50 μm slices using a vibratome (Leica). Isolated islets were processed and paraffin embedded as previously described (Lorenzo et al., 2015 (link)). RAW264.7 cells and iPCS-derived islet cells were washed with PBS and fixed 15 minutes in 4% paraformaldehyde at room temperature. They were washed again with PBS and permeabilized with PBS-0.1% triton 15 minutes on ice. Tissue sections/cells/free floating brain sections were blocked in PBS containing 5% donkey/goat serum and 0.2% Triton ×-100 for 1h at R/T. Immunostaining was then performed overnight at 4°C using a combination of primary antibodies (Key resources table ). Subsequently, secondary antibodies were incubated for 1 hour at room temperature in PBS 0.2% TritonX100 (Key resources table ). Nuclei were stained with 0.0001% of 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and cover slips were mounted using fluorescent mounting medium (DAKO). Epifluorescence microscopy images were acquired with a Leica DM6000B microscope and z-stack images were acquired using a Confocal Leica TCS SP5 (AOBS) microscope.
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