After incubation with pneumolysin, cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min, and blocked with 3% bovine serum albumin (BSA) in PBS for 40 min. Primary antibodies against γH2AX (Ser-139) (Millipore #05–636), 53BP1 (Santa Cruz #sc-22760), MDC1 (Abcam #1169), p53BP1 (S1778, Cell signaling #2675) were used at 1:100 dilutions in PBS, and incubated for 1 h at room temperature with cover slips. For TUNEL staining, the labeling enzyme (Roche #1 1684795 910) was incubated similarly for 1 h at 37 °C. Secondary antibodies (Invitrogen) labeled with either Alexa Fluor 488 or 564 or 647 were used. For EdU detection, Alexa Fluor azide was used in Click-iT reaction buffer (Invitrogen). Slides were mounted with SlowFade (Invitrogen) and stored at 4 °C until imaging. All stained slides were examined under confocal microscope, and 15 images (at 5 × 3 sites) were taken for each well under 40X magnification.
Click it reaction buffer
Manufactured by Thermo Fisher Scientific
The Click-iT reaction buffer is a component used in the Click-iT reaction, a technique for labeling and detecting biomolecules such as proteins, nucleic acids, and lipids. The buffer provides the necessary chemical environment for the Click-iT reaction to occur, enabling the incorporation of labeled components into the target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Reaction buffer additive, by Thermo Fisher Scientific (2 mentions)
Slowfade, by Thermo Fisher Scientific (1 mentions)
Sc 22760, by Abcam (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Click it nascent rna capture kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Merck Group (1 mentions)
Spe confocal microscopy, by Leica (1 mentions)
Alexa fluor 647 azide, by Thermo Fisher Scientific (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
Fv3000, by Olympus (1 mentions)
4 protocols using click it reaction buffer
1
Immunofluorescence Staining of DNA Damage Markers
2
RNA Labeling and Capture Protocol
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Newly synthesized RNA was isolated using Click-iT Nascent RNA Capture Kit (Invitrogen) according to the manufacturer's instruction. Briefly, cells were incubated in 0.2 mM of 5-ethymyl uridine (EU, an alkyne-modified uridine analog, which is efficiently and naturally incorporated into the nascent RNA) for 4 h and total RNA labeled with EU was isolated using TRIzol reagent (Invitrogen). Then, EU-labeled RNA was biotinylated in a Click-iT reaction buffer (Invitrogen) with 0.5 mM of biotin azide, and subsequently captured on streptavidin magnetic beads.
3
Labeling Proliferating Cells in AC Spheres
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The tertiary AC spheres were treated with 5-ethynyl-2-deoxyuridine (EdU, 200 ng/mL, Sigma) for 48 h as previously reported [28 (link),29 (link),34 (link)]. The spheres were fixed by 4% paraformaldehyde at room temperature for 10 min, followed by incubation in Click-iT reaction buffer, CuSO4, Alexa Fluor 555, and reaction buffer additive (Invitrogen) for 30 min. All nuclei were labeled with Hoechst 33342 (nuclei marker). The sphere samples were observed and imaged using Leica SPE confocal microscopy.
4
Quantifying Newly Generated Cells in Mouse Brain
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EdU (5-ethynyl-2’-deoxyuridine) staining was used to identify newly-generated cells. The EdU (Invitrogen) was delivered to each mouse weekly at 10 mg/kg by intraperitoneal injection from 6 weeks to 8 weeks after BCAS. The mice were sacrificed 2 months after BCAS and the brain was cut into frozen slices. Then the slices were fixed in 4% paraformaldehyde in PBS for 15 min. Next, the slices were washed twice with 3% BSA and permeabilized with 0.5% Triton X-100 in PBS for 20 min. The slices were again washed twice with 3% BSA in PBS and then incubated with a cocktail containing Click-iT™ reaction buffer, CuSO4, Alexa Fluor® 647 Azide, and reaction buffer additive (Invitrogen) for 30 min. The slices were then washed twice with PBS and further immunofluorescence staining was applied before using a laser scanning confocal microscope (FV3000, Olympus). All steps were carried out at room temperature.
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