Approximately 104 HeLa cells were grown overnight in μ-Slide Angiogenesis (ibidi). PLA was conducted using the Duolink In Situ PLA Kit (Sigma-Aldrich, DUO92101) according to the manufacturer’s protocol. The primary antibodies used in Fig. 2 (A and B) and fig. S6B were as follows: rabbit polyclonal anti-RAD21 (Abcam, ab154769), mouse monoclonal anti-FUS immunoglobulin G1 (IgG1; clone 4H11; Santa Cruz Biotechnology, sc-47711), mouse monoclonal anti-HNRNP M1-4 IgG1 (clone 1D8; Santa Cruz Biotechnology, sc-20002), mouse monoclonal anti–U1-70K (clone 9C4.1; EMD Millipore, 05-1588), and mouse monoclonal anti-nucleolin (C23, clone MS-3; Santa Cruz Biotechnology, sc-8031). The primary antibodies used in Fig. 4A and fig. S11A were as follows: mouse monoclonal anti-BRD4 (Sigma-Aldrich, AMAB90841), rabbit polyclonal anti-RAD21 (Abcam, ab154769), rabbit polyclonal anti-SMC3 (Abcam, ab9263), and anti-Tbp1 (Abcam, ab63766). The primary antibodies for RAD21, U1-70, and nucleolin used in Figs. 5 and 7D and figs. S13 and S15 were the same as above; the BRD4 antibody in Fig. 5C used was clone BL-149-2H5 (Bethyl Laboratories, A700-004). Cells were analyzed using either the Zeiss LSM880 Multi-Photon Confocal Microscope or the Yokogawa spinning disk confocal/Nikon Eclipse Ti2 microscope. The signal quantification was done using ImageJ with a custom macro.
Ab154769
Manufactured by Abcam
Sourced in United Kingdom
Ab154769 is a laboratory equipment product manufactured by Abcam. It is designed for specific laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information about the product's core function and intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Las af, by Leica (2 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Sc 7292, by Abcam (2 mentions)
Alexa fluor 700 goat anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Hpx pl apo 63x 1.40 0 6 oil immersion objective, by Leica (2 mentions)
Tcs sp5 2 confocal microscope, by Leica (2 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (2 mentions)
D6c8t, by Cell Signaling Technology (2 mentions)
D9b8n, by Cell Signaling Technology (2 mentions)
Clarity western ecl substrate reagent, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Alexa fluor 647 goat anti rabbit, by Abcam (2 mentions)
Goat anti rabbit alexa fluor 568 antibodies, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Z033429 2, by Agilent Technologies (2 mentions)
Fluorsave reagent, by Merck Group (2 mentions)
Duolink in situ pla kit, by Merck Group (1 mentions)
9 protocols using ab154769
1
Proximity Ligation Assay in HeLa Cells
2
Immunofluorescence Staining of Nuclear Proteins
3
Quantifying Nuclear RAD21 Levels by Microscopy
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Sterile 13 mm coverslips were seeded with 2 × 105 cells and kept in a cell culture incubator at 37 ◦C overnight. Coverslips were washed with PBS and fixed for 5 min with 300 μL ice-cold 100% methanol at room temperature followed by three washes with PBS and stored at 4 °C overnight. Cells were permeabilised with 200 μL/sample 0.5% Triton X-100 in PBS for 10 min, incubated with 10% serum for 30 min, and primary antibody incubation was performed at 4 °C overnight using RAD21 (Abcam ab154769) 1:500 in 10% serum. Cells were washed three times with PBS and secondary antibody incubation was performed with goat α-rabbit 488 (Invitrogen A11034 1:500) for 1 h at room temperature. Confocal stacks were acquired in Leica SP8 microscope with a voxel size of 0.144 × 0.144 × 0.99 μm, ×40 oil objective). For the analysis, nuclei and cell outlines were identified from maximum projections by CellProfiler v2.2.0 (ref. 70 (link)) as primary and secondary objects. To normalise variable levels of background, RAD21 expression for each cell was defined as the mean nuclear intensity of the antibody channel minus the cytoplasmic mean. Example images were filtered to remove speckle background noise using a CellProfiler filter.
4
Immunofluorescence Staining of Nuclear Proteins
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Immunofluorescence staining was performed at room temperature. Fixed cells were permeabilized with 0.1% triton (Sigma T8787) in 1× PBS for 10 minutes. Cells were blocked with 3% BSA (Sigma A7906) in 0.1% triton/PBS for 1 hour. Cells were incubated with primary antibody diluted in the blocking buffer for 2 hours [Lamin A/C (636) mouse mAb (1:800, SantaCruz sc-7292 lot C2219), Rad21 rabbit pAb (1:1000, abcam ab154769, lot GR3224138-10), CTCF rabbit pAb (1:800, Cell Signaling 2899, lot 2)]. Cells were washed with 0.1% triton/PBS 3 × 5 minutes. Cells were incubated with secondary antibodies [goat anti-rabbit IgG H&L Alexa Fluor 488 (1:1000, abcam ab15007, lot GR3225678-1), goat anti-mouse IgG H&L Alexa Fluor 700 (1:1000, invitrogen A-21036, lot 2084419)] diluted in the blocking buffer and conjugated tubulin antibody [anti-tubulin (YOL1/34)-AlexaFluor647, rat mAb, 1:100, abcam ab195884, lot GR281429-4] for 1 hour in the dark.
Cells were washed with 0.1% triton/PBS 1 × 5 minutes and then washed with 1× PBS 3 × 5 minutes. Coverslips were mounted to slides using ProLong Diamond Antifade Mountant with DAPI (Invitrogen P36962). For image acquisition, we used a Leica TCS SP5- II confocal microscope with 405 nm, 488 nm, 561 nm, and 633 nm lasers. Imaging we performed using a Leica HPX PL APO 63X/1.40-0.6 oil immersion objective with standard PMTs. Images were acquired using Leica LAS AF.
Cells were washed with 0.1% triton/PBS 1 × 5 minutes and then washed with 1× PBS 3 × 5 minutes. Coverslips were mounted to slides using ProLong Diamond Antifade Mountant with DAPI (Invitrogen P36962). For image acquisition, we used a Leica TCS SP5- II confocal microscope with 405 nm, 488 nm, 561 nm, and 633 nm lasers. Imaging we performed using a Leica HPX PL APO 63X/1.40-0.6 oil immersion objective with standard PMTs. Images were acquired using Leica LAS AF.
5
Immunoblotting of Pluripotency Markers
6
Visualizing Chromatin-Associated Proteins in Cells
7
Immunofluorescence Analysis of Meiotic Markers
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The primary antibodies were rabbit anti-REC8 (ab192241; Abcam, UK), rabbit anti-RAD21 (ab154769; Abcam), rabbit anti-SMC3 (ab9263; Abcam), mouse anti-OCT3/4 (sc5297; Santa Cruz Biotechnology, USA), and rabbit anti–α-tubulin (ab4074; Abcam). The secondary antibodies were AffiniPure goat anti-rabbit IgG (H+L) (111-005-003; Jackson ImmunoResearch, USA) and AffiniPure goat anti-mouse IgG (H+L) (115-005-003; Jackson ImmunoResearch).
8
Immunoblotting of Pluripotency Markers
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mESCs and NPCs were harvested and lysed in RIPA lysis buffer (150 mMNaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 25 mM Tris (pH = 7.4)). The 6% in-house made SDS-PAGE gels were used to separate the WAPL and RAD21 proteins, and the 10% SDS-PAGE gels was used for SOX2, OCT4 and NANOG. The separated protein was transferred to a pre-activated PVDF membrane using Trans-Blot Turbo Transfer System (Bio-Rad). The blots were incubated with the following primary antibodies overnight at 4°C: (1) WAPL (1:1,000, 16370-1-AP, Proteintech), (2) RAD21 (1:1,000, ab154769, Abcam), (3) SOX2 (1:1,000, D9B8N, Cell Signaling), (4) OCT4 (1:1,000, D6C8T, Cell Signaling), (5) NANOG (1:1,000, D2A3, Cell Signaling), and (6) HSP90 (1:2,000, 13171-1-AP). After incubation, the blots were washed 3 times with TBS-0.1% Tween-20. The blots were then incubated with secondary antibody against rabbit IgG at room temperature for 1 h, following by 3-time TBS-0.1% Tween-20 washing. The proteins attached with antibodies were hybridized with Clarity Western ECL Substrate reagent (Bio-Rad) and visualized in a ChemiDoc MP Imaging System (Bio-Rad).
9
Visualizing Chromatin-Associated Proteins in Cells
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For GFP visualization cells were grown on poly-L-lysine (Sigma Aldrich) coated chamber slides (ThermoFisher Scientific), fixed in 4% formaldehyde (FA) and nuclei were counterstained with Hoechst 33342 (ThermoFisher Scientific).
For RAD21 immunofluorescence analysis in mESCs, we let single cells adhere for 30 min on poly-L-lysine coated slides. Next, pre-extraction of the non-chromatin-associated RAD21 fraction was performed by incubation with 0.1% Triton X-100 in PBS for 1 min followed by fixation with 4% FA. Staining was performed with rabbitanti-RAD21 (Abcam, ab154769, 1:200) followed by incubation with goat anti-rabbit Alexa Fluor 647 (Abcam, 1:250). Nuclei were counterstained with 4’,6-Diamidino-2-Phenylindole (DAPI) (ThermoFisher Scientific). For NPCs, cells were grown on poly-L-lysine coated coverslips fixed in 4% FA and stained with mouse anti-NESTIN (BD biosciences, 611659, 1:200) and rabbit anti-GFAP (DAKO, Z033429-2, 1:100) antibodies, followed by incubation with goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 568 antibodies (both ThermoFisher Scientific, 1:250). Nuclei were counterstained with DAPI. Prior to imaging all samples were mounted with FluorSave reagent (Merck). Fluorescent confocal images were captured on a Leica SP5 system (Leica, Wetzlar, Germany). All the immunofluorescence images were processed using ImageJ software (version 1.53c).
For RAD21 immunofluorescence analysis in mESCs, we let single cells adhere for 30 min on poly-L-lysine coated slides. Next, pre-extraction of the non-chromatin-associated RAD21 fraction was performed by incubation with 0.1% Triton X-100 in PBS for 1 min followed by fixation with 4% FA. Staining was performed with rabbitanti-RAD21 (Abcam, ab154769, 1:200) followed by incubation with goat anti-rabbit Alexa Fluor 647 (Abcam, 1:250). Nuclei were counterstained with 4’,6-Diamidino-2-Phenylindole (DAPI) (ThermoFisher Scientific). For NPCs, cells were grown on poly-L-lysine coated coverslips fixed in 4% FA and stained with mouse anti-NESTIN (BD biosciences, 611659, 1:200) and rabbit anti-GFAP (DAKO, Z033429-2, 1:100) antibodies, followed by incubation with goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 568 antibodies (both ThermoFisher Scientific, 1:250). Nuclei were counterstained with DAPI. Prior to imaging all samples were mounted with FluorSave reagent (Merck). Fluorescent confocal images were captured on a Leica SP5 system (Leica, Wetzlar, Germany). All the immunofluorescence images were processed using ImageJ software (version 1.53c).
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