DSB repair foci were analyzed using co-staining of γH2AX and 53BP1, as described previously (32 (link)). Cells were fixed and stained at 0, 4, and 24 h after irradiation with a primary antibody solution as follows: mouse monoclonal anti-phospho-S139-H2AX antibody (1:500, clone JBW301, Millipore, Darmstadt, Germany) and rabbit polyclonal 53BP1 antibody (1:500, Novus Biologicals, Wiesbaden, Germany). Secondary antibody solutions were mouse Alexa-Fluor 594 (1:1,000) and rabbit Alexa-Fluor 488 (1:1,000, both Invitrogen, Karlsruhe, Germany). Cells were mounted in ProLong Gold Antifade Reagent with DAPI (Invitrogen, Karlsruhe, Germany). Immunofluorescence was analyzed using the Leica DM5500 wide-field microscope and LAS-AF software (Leica, Wetzlar, Germany). All experiments were performed at least twice and with 100 counted nuclei per experiment.
Dm5500 widefield microscope
Manufactured by Leica
Sourced in Germany
The Leica DM5500 widefield microscope is a versatile and advanced imaging system designed for scientific research and analysis. It features a high-performance optical system that delivers exceptional image quality and resolution. The microscope is equipped with a wide range of illumination options, including LED and halogen, allowing for flexible and efficient sample observation. The DM5500 is capable of supporting a variety of sample types and can be configured with a range of objectives to suit the specific needs of the user.
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Lab products found in correlation
Ab49873, by Abcam (2 mentions)
Rnascope multiplex fluorescent v1 assay, by Advanced Cell Diagnostics (2 mentions)
Mm per1, by Advanced Cell Diagnostics (2 mentions)
Tissue tek, by Sakura Finetek (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Rabbit alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Mouse alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Las af software, by Leica (1 mentions)
Mouse monoclonal anti phospho s139 h2ax antibody, by Merck Group (1 mentions)
53bp1 antibody, by Novus Biologicals (1 mentions)
4 6 diamidin 2 phenylindol dapi, by Vector Laboratories (1 mentions)
Fluorescently labelled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Periodic acid solution, by Merck Group (1 mentions)
Schiff s reagent, by Merck Group (1 mentions)
7 protocols using dm5500 widefield microscope
1
Quantification of DNA Double-Strand Break Repair
2
Zonal Expression of Clock Genes in Liver
3
Zonal Expression of Clock Genes in Liver
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smFISH of R genes were done on fresh-frozen liver cryosections (8μm) embedded in O.C.T Compound (Tissue-Tek; Sakura-Finetek USA), sampled every three hours (ZT0 to ZT21). RNAscope® probes for Bma1l mRNA (Mm-Arntl, catalog #: 438748-C3) and Per1 mRNA (Mm-Per1, catalog #: 438751) were used, according to the manufacturer’s instructions for the RNAscope Fluorescent Multiplex V1 Assay (Advanced Cell Diagnostics). To detect the central vein, an immunofluorescence of Glutamine Synthetase (ab49873, Abcam, diluted 1:2000 in PBS/BSA 0.5%/Triton-X0.01%) was done together with smFISH. Nuclei were counterstained with DAPI and sections were mounted with ProLong™ Gold Antifade Mountant. Liver sections were imaged with a Leica DM5500 widefield microscope and an oil-immersion x63 objective. Z-stacks were acquired (0.2μm between each Z position) and mRNA transcripts were quantified using ImageJ, as described previously in ref.50 (link). Pericentral (PC) and Periportal (PP) veins were manually detected based on Glutamine Synthetase IF or on bile ducts (DAPI staining). The Euclidean distance between two veins and the distance from the vein of each mRNA transcript were calculated. mRNA transcripts were assigned to a PP or PC zone if the distance from the corresponding vein was smaller than one-third of the distance between the PP and PC veins (ranging from 50 to 130μm).
4
Immunofluorescence Staining of Acetylated Tubulin
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Cells were seeded on etched cover slips and treated according to the respective experimental protocol. Subsequently, cells were fixed in 4% formaldehyde/PBS (10 min at room temperature (RT), washed, permeabilized with 0.5% Triton-X100/PBS at RT for 5 min, and blocked with 10% serum/PBS for 1 h at RT. Then, cover slips were incubated with the primary antibody (Acetylated tubulin (AcTub); Sigma #T6793) in antibody-solution (PBS containing 10% serum and 0.1% Saponin) overnight at 4 °C. After washing with PBS at RT, cover slips were incubated with fluorophore-coupled secondary antibodies diluted in antibody-solution at RT in the dark for 2 h. After washing with PBS and rinsing with water, cover slips were mounted with mounting medium containing 4′,6-Dia (midin-2-phenylindol (DAPI) (Vectashield). Microscopy was performed on a Leica DM 5500 Wide field microscope (Leica Microsystems, Wetzlar, Germany) using 3D deconvolution. Resulting z-stacks are presented as maximum intensity projections.
5
Immunofluorescence Staining of FFPE Tissue
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Formalin-fixed and paraffin-embedded tissue were sectioned, de-paraffinized and antigens were retrieved using boiling 10 mM citrate (pH 6). Subsequently, sections were permeabilized with 0.2% Triton X100/PBS (5 min, RT) and blocked with 10% serum/PBS (1 h, RT). Primary antibody staining was overnight (4 °C) using anti-acetylated α-tubulin (Sigma T6793) and anti-pericentriolar material 1 (PCM1; CST #5213) antibodies, followed by a 1–2 h incubation at RT with fluorescently labelled secondary antibodies (Invitrogen). Sections were embedded in Vectashield/DAPI and visualized on a Leica DM 5500 Wide field microscope (Leica Microsystems, Wetzlar, Germany) using 3D deconvolution. Resulting z-stacks are presented as maximum intensity projections. The use of human patient material through the Marburg Biobank was approved by the local ethics committee at the University Hospital Marburg.
6
Tissue Preparation and FISH Analysis
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Colonic intestinal tissues containing fecal pellets were fixed overnight with methacarn at room temperature. Tissues were washed in methanol for 30 min, two times in ethanol for 15 min, once in ethanol/xylene (1:1) for 15 min, and two times in xylene for 15 min prior to paraffin embedding.
For FISH analysis, 3–5 μm thin tissue sections were dewaxed and then treated with 50 μl 4% lysozyme solution (45 min, 37 °C) for nucleic acids demasking. After washing, 50 μl hybridization solution (0.9M NaCL, 20 mM Tris-HCL, 0.05% SDS) with the bacterial probe (1:50 Eub338 FITC-GCT GCC TCC CGT AGG AGT) was added and incubated for 3 h at 50 °C. Slides were washed several times at 37 °C and were dried at RT before mounted with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) following manufacturer’s instructions. The samples were documented at a Leica DM5500 wide-field microscope (Leica) and analyzed with Imaris software (Bitplane).
For FISH analysis, 3–5 μm thin tissue sections were dewaxed and then treated with 50 μl 4% lysozyme solution (45 min, 37 °C) for nucleic acids demasking. After washing, 50 μl hybridization solution (0.9M NaCL, 20 mM Tris-HCL, 0.05% SDS) with the bacterial probe (1:50 Eub338 FITC-GCT GCC TCC CGT AGG AGT) was added and incubated for 3 h at 50 °C. Slides were washed several times at 37 °C and were dried at RT before mounted with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) following manufacturer’s instructions. The samples were documented at a Leica DM5500 wide-field microscope (Leica) and analyzed with Imaris software (Bitplane).
7
Preservation of Intestinal Mucus for Histological Analysis
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The water-free methacarn (methanol-Carnoy’s) fixation was used to preserve the intestinal mucus layer during histological preparation.67 (link) Intestinal tissues with fecal pellets were fixed over night with methacarn at room temperature. Tissues were washed in methanol for 30 min, two times in ethanol for 15 min, once in ethanol/xylene (1:1) for 15 min and two times in xylene for 15 min prior to embedding in paraffin.
For periodic-acid-Schiff (PAS) histology, 3–5 µm thin tissue sections were dewaxed and stained for 10 min with periodic acid solution (0.5%, w/v; Merck), 20–30 min in Schiff’s reagent (Merck) and 1 min in Mayer’s Hämalaun (Carl Roth). Between each step, sections were rinsed in running water.
For FISH analysis, 3–5 µm thin tissue sections were dewaxed and then treated with 50 µl 4% lysozyme solution (45 min, 37°C) to demask nucleic acids. After washing, 50 µl hybridization solution was added and incubated for 3 hours at 50°C. Slides were washed several times at 37°C and were dried at RT before mounted with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) following manufacturer’s instructions. The samples were documented at a Leica DM 5500 wide field microscope (Leica) and analyzed with ImageJ (National Institutes of Health).
For periodic-acid-Schiff (PAS) histology, 3–5 µm thin tissue sections were dewaxed and stained for 10 min with periodic acid solution (0.5%, w/v; Merck), 20–30 min in Schiff’s reagent (Merck) and 1 min in Mayer’s Hämalaun (Carl Roth). Between each step, sections were rinsed in running water.
For FISH analysis, 3–5 µm thin tissue sections were dewaxed and then treated with 50 µl 4% lysozyme solution (45 min, 37°C) to demask nucleic acids. After washing, 50 µl hybridization solution was added and incubated for 3 hours at 50°C. Slides were washed several times at 37°C and were dried at RT before mounted with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) following manufacturer’s instructions. The samples were documented at a Leica DM 5500 wide field microscope (Leica) and analyzed with ImageJ (National Institutes of Health).
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