After irradiation and incubation for 1 h at 37°C, cells were permeabilized with 0.5% Triton X and blocked and stained with anti‐γH2AX antibody (ab22551; Abcam) at 1:800 dilution. Cy3 conjugated donkey anti mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as a secondary antibody, at 1:500 dilution for 1 h, and mounted with Prolong Gold with DAPI (Life Technologies). All the images were captured by a Leica DMLB fluorescent microscope (Leica Microsystems, Wetzlar, Germany). The mean density of γH2AX expression per nuclei were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Dmlb fluorescent microscope
Manufactured by Leica
Sourced in Germany
The DMLB fluorescent microscope is a laboratory equipment designed for fluorescence microscopy applications. It provides essential functionality for visualizing and analyzing fluorescently labeled samples. The DMLB microscope offers high-quality optics and illumination to enable clear and detailed observations.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal, by Leica (2 mentions)
Cy3 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions)
Ab22551, by Abcam (1 mentions)
Stereoinvestigator, by MBF Biosciences (1 mentions)
81 confocal microscope, by Olympus (1 mentions)
Fluorview fv1000, by Olympus (1 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
Hep 2 slides, by Bio-Rad (1 mentions)
α sma ab5694, by Abcam (1 mentions)
Cm1900 cryostat, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Mouse igg1 mab002, by R&D Systems (1 mentions)
Mouse igg1κ 400101, by BioLegend (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Triton x, by Thermo Fisher Scientific (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole dilactate, by Thermo Fisher Scientific (1 mentions)
Fluorescent mounting media, by Agilent Technologies (1 mentions)
10 protocols using dmlb fluorescent microscope
1
Quantifying DNA Damage Response
2
Quantifying Brain Metastasis Distribution
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Two cohorts consisting of six to eight male NSG mice each were utilized to quantify the distribution of metastasis in different brain regions. One cohort of animals was subjected to stroke and metastasis and the other with metastasis alone. Brains were extracted, fixed in 4% paraformaldehyde and cryosectioned into 100 μm thick sagittal sections while frozen in OCT. Four sections per animal were used to quantify metastasis and the distribution of YDFR-CB3GFP+ cells in various brain regions. Sections were imaged, outlined, and GFP+ cells were manually counted using a StereoInvestigator (StereoInvestigator MBF Bioscience, Williston, VT, United States) on a Leica DMLB fluorescent microscope (Leica Microsystems, Wetzlar, Germany) with a 40× objective.
3
Fluorescence Microscopy Imaging Protocol
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Single stained cells were viewed with a Leica DMLB fluorescent microscope (Leica Microsystems, Sydney, Australia) and photos taken using a SPOT camera (RTKE Diagnostic Instruments Inx, MI, USA). Double stained cells were observed under the Olympus 1x81 confocal microscope and Images acquired using the Olympus Fluorview FV1000 (Olympus Australia Pty Ltd, VIC, Australia).
4
CEACAM1 Binding Specificity of scFv DIATHIS1
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To test the scFv DIATHIS1 specificity against CEACAM1 a competitive assay was performed. For this purpose 2.5×105 MelC cells were grown overnight on glass coverlips, washed and fixed with 4% (vol/vol) of formaldehyde for 15 minutes at RT and incubated in PBS containing 1% (wt/vol) BSA and 0.1% (wt/vol) gelatin to saturate nonspecific binding sites. The cells were then exposed to 10 μg/mL of scFv DIATHIS1 alone or added in separated samples with different concentrations (5, 10, and 50 μg/mL) of soluble recombinant CEACAM1 fragment corresponding to the N-terminal IgV-like domain and first IgC2-like domain (CEACAM1 amino acid residues 35-233; Diatheva S.R.L., Fano, Italy) and then incubated for 60 minutes at RT. After washing, an anti-Flag M2 monoclonal antibody (Sigma Aldrich) 1:400 (vol/vol) diluted in blocking solution was added and incubated for 60 minutes at RT. After washing, an anti-mouse antibody FITC-conjugated 1:200 (vol/vol) diluted in blocking solution was added to the wells and maintained for 60 minutes RT. After washing, the fluorescence was highlighted using a Leica DMLB fluorescent microscope equipped with a DC300F CCD digital camera. Nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI) solution 1:10000 (vol/vol) diluted.
5
ANA Detection by Indirect Immunofluorescence
6
Multicolor Immunofluorescence Staining of Tissue
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Frozen samples, processed immediately after tissue harvesting, were cut in a Leica CM1900 cryostat. For staining, tissues were fixed in ice-cold acetone:methanol (50:50) and antibodies were prepared in antibody diluent (003118; Invitrogen-Thermo Fisher Scientific, Paisley, UK). After staining, slides were mounted in Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI;F6057; Sigma, St Louis, MO, USA). Primary antibodies used were CD146-FITC (MCA2141F; AbD Serotec-BioRad, Kidlington, UK), CD144 (AHP628Z; AbD Serotec-BioRad), NG2 (MAB2585; R&D Systems, Minneapolis, MN, USA), αSMA (ab5694; AbCam, Cambridge, UK), CD29 (303015; BioLegend, San Diego, CA, USA), CD44 (AbD Serotec-BioRad), and CD34 (21270341S; ImmunoTools, Friesoythe, Germany). Isotype controls were IgG1-FITC (MCA928F; AbD Serotec-BioRad), IgG1 (MAB002; R&D Systems), IgG1κ (400101; BioLegend), and IgG1 (21335011; ImmunoTools), all raised in mouse; and rabbit IgG (PRABP01; AbD Serotec-BioRad). Secondary antibodies were conjugated to AF488 (A11029), AF568 (A110037), and AF568 (A10042), all from Invitrogen-Thermo Fisher Scientific. Micrographs were produced using a Zeiss LSM710 confocal or Leica DMLB fluorescent microscope.
7
Immunofluorescence Staining of Tissue Samples
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Samples were processed in a Leica CM1900 cryostat, allowed to air dry, and processed right away or kept frozen at −80°C. For staining, tissues were fixed in ice cold acetone–methanol (50:50) and antibodies were prepared in diluent (003118; Invitrogen-Thermo Fisher Scientific, Paisley, United Kingdom) and, after staining, slides were mounted in fluoroshield with DAPI (Sigma-Aldrich, St Louis, MO). Primary antibodies used in the study are listed in Table 1 and isotype controls were mouse IgG1κ 400101 (BioLegend, San Diego, CA), mouse IgG1 MCA928F (AbD Serotec-BioRad, Kidlington, United Kingdom), mouse IgG1 MAB002 (R&D Systems, Minneapolis, MN), mouse IgG1κ 557273 (BD, Oxford, United Kingdom), and rabbit PRABP01 (AbD Serotec-BioRad). Secondary antibodies were AF488 conjugated (A11008 and A11029) and AF568 conjugated (A110037 and A10042). Isotypes and secondary antibodies alone were used as controls. Micrographs were produced using a Zeiss LSM710 confocal or Leica DMLB fluorescent microscope.
8
Immunofluorescence Assay for Parasite Visualization
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Immunofluorescence assays (IFA) were performed as previously described [21] (link). Briefly, HFF monolayers grown on coverslips were inoculated with the designated parasite line, sometimes containing Shield-1 or EtOH vehicle. After removal of culture medium, infected HFFs were fixed in 3% paraformaldehyde for 10 min and then were permeabilized with 0.3% Triton X-100 for 10 min. For visualization of HA-fusion proteins, rat monoclonal anti-HA primary antibody (Roche #11867423001) was applied at 1∶2,000 followed by goat anti-rat Alexa Fluor 488 secondary antibody at 1∶2,000 (Invitrogen #A-11006). Nuclei were co-stained with 4′,6-diamidino-2-phenylindole (DAPI). Samples were visualized using a Leica DMLB fluorescent microscope.
9
Immunofluorescence Analysis of p53 Expression
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D17, Abrams, and Dharma cells were seeded on 22 mm borosilicate glass coverslips (Thermo Fisher Scientific), fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, Pennsylvania), permeabilized with 0.33% Triton‐X (Thermo Fisher Scientific) in phosphate‐buffered saline (PBS), and blocked with 5% donkey serum (Sigma‐Aldrich) in PBS. Cells were incubated with wt‐p53 (Bioss) diluted 1:200 in PBS, blotted on parafilm in a humidified chamber at 4°C. Alexa‐488‐conjugated donkey anti‐rabbit IgG (H + L) (Thermo Fisher Scientific) was used as the secondary antibody (1:800, 1 hour at RT). Cells were subsequently counterstained with 300 nM 4′,6‐diamidino‐2‐phenylindole dilactate (Thermo Fisher Scientific) for 10 minutes. All cover slips were mounted with DAKO fluorescent mounting medium (Agilent Technologies, Santa Clara, California). Slides were imaged with the Leica (Wetzlar, Germany) DMLB fluorescent microscope using the 100x/1.3 aperture oil immersion lens or 10x/0.7 aperture lens for quantification with ImageJ/Fiji. An omission control was used to control for binding of the secondary antibody.
10
Immunostaining of Theileria annulata
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Cells were pelleted on to glass slides using the Cytospin system (Thermo Scientific, Paisley, UK) and fixed in acetone. Slides were stained using the Sequenza Immunostaining Center (Thermo Scientific) using 50 µL reaction volumes with reagents diluted in PBS as follows: cells were incubated with anti-T. annulata p104 vb (1C12 [13 (link)]) followed by goat anti-mouse IgG FITC conjugated antibody (Molecular Probes) then 3 nM DAPI. Slides were mounted using Fluorescent Mounting Media (Agilent Technologies, Wokingham, UK) and examined using a DMLB Fluorescent Microscope (Leica, Wetzlar, Germany).
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