HCT116 cells were plated onto poly-d -lysine-coated glass-bottom plates (Greiner BioOne, Monroe, NC) at 20,000 cells per well. The cells were then treated with or without 10 μM Omomyc for 24 h, fixed with cold methanol (Thermo-Fisher), and then permeabilized using PBS (Thermo-Fisher) containing 1% BSA and 0.1% Triton X-100. Next, the cells were blocked in PBS-BSA-Triton X-100 buffer with 0.1% horse serum (Sigma) added. Blocked cells were treated with primary antibodies to UBF (Sigma), histone H3 (Active Motif), and Max (Sigma) at 1:500 dilutions for all antibodies. Cells were washed and then stained with goat anti-rabbit IgG Atto488 (Sigma) or goat anti-mouse IgG Alexa 568 (Thermo-Fisher) secondary antibody at a 1:1,000 dilution as well as the nuclear dye Hoechst 33342 at a 1:5,000 dilution. Cells were visualized using a Nikon SIM-E superresolution microscope with a 100× objective.
Goat anti rabbit igg atto 488
Manufactured by Merck Group
Sourced in United States
Goat anti-rabbit IgG-Atto 488 is a secondary antibody conjugated with the Atto 488 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Pab12506, by Abnova (2 mentions)
Cellinsight cx5 high content screening platform, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Horse serum, by Merck Group (1 mentions)
Goat anti mouse igg alexa 568, by Thermo Fisher Scientific (1 mentions)
Sim e superresolution microscope, by Nikon (1 mentions)
Histone h3, by Active Motif (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
B0556, by Abcam (1 mentions)
Ab4566, by Abcam (1 mentions)
Ab17677, by Abcam (1 mentions)
Ab18208, by Abcam (1 mentions)
Ab24834, by Abcam (1 mentions)
Ab31830, by Abcam (1 mentions)
Anti h3, by Active Motif (1 mentions)
Anti h2a, by Merck Group (1 mentions)
Hmgn2, by Cell Signaling Technology (1 mentions)
Atto 488 goat anti mouse igg, by Merck Group (1 mentions)
5 protocols using goat anti rabbit igg atto 488
1
Immunofluorescence Analysis of HCT116 Cells
2
Immunostaining of 53BP1 DNA Damage Foci
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For the 53BP1 immunostaining experiment, 5000 cells/well were seeded in a black, transparent bottom 96-well plate. After incubation for 24 h with digested and non-digested samples, cells were fixed with 4% formaldehyde, pH 7.4, permeabilized with 0.2% Triton X-100 and washed three times with PBS containing 3% Bovine Serum Albumine (BSA) (washing buffer). They were then exposed to anti-53BP1 antibody (Abnova, PAB12506, dilution 1/1000, Taipei, Taiwan) for 1 h at room temperature under mild agitation, rinsed three times for 5 min with washing buffer, and incubated for 1 h at room temperature with goat anti-rabbit IgG-Atto 488 (Sigma Aldrich 18772, dilution 1/2000, St. Louis, MO, USA). They were further rinsed three times in washing buffer and counterstained with 0.3 µg/mL Hoechst 33,342 for 20 min at room temperature. Cells were finally washed three times with PBS, and plates were stored at 4 °C in the dark until analysis using a CellInsight CX5 High Content Screening platform (Thermo Fisher Scientific, Waltham, MA, USA), as reported previously [35 (link)].
3
Immunofluorescent Detection of DNA Damage Response Proteins
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After incubation for 24 h with TiO2 NPs, cells were fixed with 4% formaldehyde, pH 7.4, permeabilized with 0.2% triton X-100 and washed three times with PBS containing 4% non-fat dry milk (washing buffer). They were then exposed to anti-53BP1 antibody (Abnova, PAB12506, dilution 1/500, Taipei, Taiwan) for 1 h at room temperature under mild agitation, rinsed three times for 5 min with washing buffer and incubated for 1 h at room temperature with goat anti-rabbit IgG-Atto 488 (Sigma-Aldrich, 18,772, dilution 1/2000, St. Louis, MO, USA). They were further rinsed three times in washing buffer and counterstained with 5 µg/mL Hoechst 33,342 for 15 min at room temperature. Cells were finally washed three times with PBS, and plates were stored at 4 °C in the dark until analysis using a CellInsight CX5 High Content Screening platform (Thermo Fisher Scientific).
4
Immunofluorescence Staining and STED Microscopy
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All cells were were fixed with 4% PFA for 30 min, followed by permeabilizing with 0.5% Triton X-100/PBS for 20 min at RT. The cells were blocked in a blocking buffer (3% BSA, 2%donkey serum in PBS) for 10 min. Subsequently, the cells were stained with primary antibody against FBL (Abcam, ab4566;1:200), NPM (Sigam, B0556; 1:200 and Abcam, ab183340,1:100), LIN28A (CST, 3978 S; 1:200) overnight at 4 °C. The next day, they were washed three times for 10 min with PBS, and the cells were stained with a secondary antibody (Anti-mouse IgG Alexa Fluor® 647 Conjugate, Jackson ImmunoResearch, 115-605-003,1:400; atto 488-goat anti-rabbit IgG, Sigma, 18772, 1:150) for 1 h at 37 °C. Following washing three times with PBS, DAPI was used for nucleus staining. STED images were acquired using Abberior Instruments with z-stack module. The x, y, and z axis resolution was 30 nm. STED Resol. was 5%. The images were analyzed using Fiji/ImageJ. In order to quantify nucleolus regularity, Boyce-Clark index was used37 (link): ri is the length from the vantage center to the boundary (semidiameter) of the nucleolus, n is the number of the semidiameter. FBL regularity data were up to the nearest integer in Fig. 7g .
5
Antibody Immunostaining Protocol
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The following primary rabbit antibodies were purchased from Abcam: anti-H1.2, ab17677; H1.5, ab18208; HMGB2, ab124670; Ki67, ab15580; H3S10p, ab5176; CTCF, ab128873; SMC2, ab10412; RAD21, ab154769; H1x, ab31972. From Cell Signaling: anti-HMGN1, #5692; HMGN2, #9437. From Sigma-Aldrich: anti-H1.4, H7665. From Millipore: anti-H2A, #07-146. The following mouse antibodies were purchased from Abcam: anti-H3, ab24834; H4, ab31830; H2B, ab52584. From Active Motif: anti-H3, #61475. mAb PL2-6 was a gift from M. Monestier (Temple Univ.), see previous use [26 ,27 ]. Secondary antibodies included Invitrogen Alexa 568 goat anti-rabbit IgG, Sigma-Aldrich Atto 488 goat anti-rabbit IgG and Atto 488 goat anti-mouse IgG.
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