Cells were seeded on 13 mm glass coverslips (VWR, 631-0148) while reverse transfected when applicable. The samples were washed once in ice-cold PBS and fixed with 4% formaldehyde in PBS for 15 min at RT. When pre-extraction was performed in order to remove the non-chromatin-bound fraction, this was carried out prior to fixation using 0.2% Triton in PBS for 90 sec. The samples were permeabilized in 0.5% Triton in PBS for 15 min prior to incubation with primary antibodies for 1–2 h depending on the antibody. The samples were subsequently incubated with secondary fluorescence-coupled antibodies (Alexa Fluor) for 1 h and finally stained with DAPI at 1 μg/ml for 5 min before being mounted on glass slides with Prolong Diamond mounting medium (Invitrogen, P36961). The coverslips were washed at least 3 times after each antibody incubation as well as after DAPI staining. Incubations with antibodies were done in 3% BSA in PBS-T (0.01% Tween-20 in PBS) and all washes were performed with PBS-T. When Click-it reactions were performed as for EdU incorporation, this was done prior to the primary antibody incubation according to the manufacturer's instructions (Invitrogen, C10638).
C10638
Manufactured by Thermo Fisher Scientific
The C10638 is a laboratory instrument manufactured by Thermo Fisher Scientific. It is a core piece of equipment designed for general laboratory use. The device's primary function is to perform specific laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
C10337, by Thermo Fisher Scientific (2 mentions)
Axio imager m2, by Zeiss (2 mentions)
Prolong diamond mounting medium, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Anti foxp3, by Thermo Fisher Scientific (1 mentions)
Anti nras, by Santa Cruz Biotechnology (1 mentions)
Fluorochrome tagged secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Anti cox 2, by Santa Cruz Biotechnology (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
Anti ki67, by Fortis Life Sciences (1 mentions)
Anti ly6c, by Abcam (1 mentions)
Anti cd11c, by Cell Signaling Technology (1 mentions)
Anti cxcl1, by Abcam (1 mentions)
Anti pge2, by Abcam (1 mentions)
Anti p21, by BD (1 mentions)
6 protocols using c10638
1
Immunofluorescence Staining Protocol
2
Quantifying DNA Synthesis in NSCLC Cells
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Newly synthesized DNA in NSCLC cells was detected by the EdU fluorescence staining (C10638, Invitrogen) according to the manufacturer’s directions. Cells were visualized by the Olympus FV1000 confocal microscope (Olympus, Japan). The percentage of EdU-positive cells was shown as the ratio between the number of EdU-stained cells (red) and the total number of Hoechst 33342-stained cells (blue) counted × 100%.
3
Immunohistochemical Profiling of Mouse Tissues
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Immunohistochemistry was performed as reported previously (Hoare et al., 2016 (link)). Formalin fixed paraffin-embedded mouse tissues were stained with the following antibodies: anti-Cox2 (as above); anti-NRAS (Santa Cruz, sc-31,1:100); anti-p21 (BD, 556431, 1:50); anti-ki67 (Bethyl, IHC-00375, 1:1000); anti-Ly6C (Abcam, ab15627, 1:400); anti-Cd11c (Cell Signaling, 97585, 1:350); anti-Cxcl1 (Abcam, ab86436, 1:100); anti-PGE2 (Abcam, ab2318, 1:100); anti-Foxp3 (eBioscience, 14-5773, 1:100); after proteinase K digestion (Ly6C) or heat-induced epitope retrieval in citrate (pH6) or Tris-EDTA (pH9) buffers before visualization using the DAKO Envision kit according to manufacturer’s instructions and counterstaining with hematoxylin. For fluorescent labeling we utilized appropriate fluorochrome-tagged secondary antibodies (Life Technologies). For EdU staining (ThermoFisher C10638), the same protocol was followed, with the following extra step: after antigen retrieval, 3% BSA washes in PBS were performed twice, and CliCK-iT reaction cocktail was added for 30 min following the manufacturer’s instructions.
All slides were scanned on a Leica AT2 at 20x magnification and a resolution of 0.5 μm/pixel. Following digitization, image analysis was performed as described previously using HALO (Indicalabs) (Hoare et al., 2016 (link)).
All slides were scanned on a Leica AT2 at 20x magnification and a resolution of 0.5 μm/pixel. Following digitization, image analysis was performed as described previously using HALO (Indicalabs) (Hoare et al., 2016 (link)).
4
Astrocyte Proliferation Quantification
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P5 pups were Intraperitoneally injected with 100 mg/Kg EdU (Thermo Fisher Scientific, C10337 or C10638) and collected at P7. Brain tissue was processed as described above. After antigen retrieval, sections were washed with 10% goat serum in PBS for 5 minutes and applied Click-iT solution as described in the kit. After 30 minutes EdU staining, sections were washed with 10% goat serum in PBS for 5 minutes, then proceed to immunostaining with desired markers. Images were acquired using Zeiss Axio Imager.M2 with apotome and 20X objective. To quantify proliferating astrocytes, colocalization of EdU and astrocyte markers were analyzed by QuPath software.
5
Chlamydia Infection Dynamics in Cervical Epithelial Cells
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A2EN human cervical epithelial cells were seeded at 1 × 105 on coverslips, grown overnight, and then exposed to starvation medium (EpiLife medium) for 18 h. Coverslips were exposed to either EpiLife complete medium (control) or CDK4 inhibitor CAS 546102-60-7 at 400 nM (Millipore 219476) or Fascaplysin at 350 nM (Millipore 341251) in complete medium. Concurrently, C. trachomatis serovar L2 was added at an MOI of 2 to each coverslip and rocked for 2 h at room temperature. A2EN cells were then exposed to complete medium or inhibitors in the presence of EdU (1 nM) as described above for an additional 22 h at 37°C in 5% CO2. Cells were fixed, stained, and imaged for C. trachomatis , EdU (Alexa Fluor 555; ThermoFisher C10638), or cell viability staining as outlined above. The proportion of infected A2EN cells and new DNA synthesis were determined using manual counting and CellProfiler software. A two-tailed t test was applied to test whether differences between means of each EdU or infection experimental conditions were equal to 0, with P < 0.05 considered significant. A linear model was fitted to mean percent cells stained with EdU versus the mean proportion of infected A2EN cells using the R lm package (version 3.2.3).
6
Astrocyte Proliferation Quantification
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