MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked with 5% BSA in PBS for 1 h. A rabbit anti-RNF187 (HPA030098, Sigma, 1:100) antibody and mouse anti-P53 monoclonal antibody (SC126, Santa Cruz, 1:100) were used as primary antibodies, followed by Alexa Fluor 647-conjugated (Invitrogen) anti-rabbit and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA) as secondary antibodies. As negative controls, samples were incubated with secondary antibodies without the primary antibody incubation step. Images were acquired under conditions satisfying the Nyquist criterion using a Nikon A + laser scanning confocal microscope system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy unit. The acquired images were further processed and assembled using ImageJ.
Oil na1.4 objective
Manufactured by Nikon
The 60X oil NA1.4 objective is a high-magnification, high-numerical aperture objective lens designed for use in laboratory equipment. It provides a 60X magnification and a numerical aperture of 1.4, which allows for high-resolution imaging and light collection. The objective is designed to be used with oil immersion, which enhances its optical performance. This objective is suitable for a variety of laboratory applications that require high-resolution imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated anti mouse antibody, by Jackson ImmunoResearch (12 mentions)
Alexa flour 647, by Thermo Fisher Scientific (10 mentions)
Hap043396, by Merck Group (4 mentions)
Sc 126, by Santa Cruz Biotechnology (3 mentions)
Hpa030098, by Merck Group (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Sc 101199, by Santa Cruz Biotechnology (1 mentions)
Hap030098, by Merck Group (1 mentions)
Hap035000, by Merck Group (1 mentions)
12 protocols using oil na1.4 objective
1
Immunofluorescent Localization of RNF187 and p53
2
Immunofluorescence Analysis of USP1 and ERα
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MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit Anti-USP1 (SAB1406575, Sigma) rabbit antibody and mouse anti-ERα monoclonal antibody (SC-56833) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
3
Immunofluorescent Localization of TRIM3 and P53
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MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-TRIM3 polyclonal antibody (HAP043396, Sigma) and mouse anti-P53 monoclonal antibodies (SC-126, Santa Cruz) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A + laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
4
Immunofluorescence Assay for TRIM3 and ERα
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MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min and blocked with 5% BSA in PBS for 1 h. Rabbit anti-TRIM3 (HAP043396, Sigma, 1:200) and mouse anti-ERα monoclonal antibodies (SC-56833, 1:200) were used, followed by an Alexa Fluor 647 (Invitrogen, 1:400) anti-rabbit antibody and a FITC-conjugated anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA, 1:400). As negative controls, samples were incubated with secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using a Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy units. The acquired images were further processed and assembled using ImageJ.
5
Immunofluorescence Imaging of RNF181 and YAP
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BT549 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.25% Triton X-100 for 5 min, and blocked by 3% BSA in PBS for 1 h. anti-RNF181 polyclonal antibody (SAB1401685, Sigma) and anti-YAP monoclonal antibodies (SC-101199, Santa Cruz) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A + laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
6
Immunofluorescence Staining of MCF-7 Cells
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MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit Anti-RNF181 (SAB1401685, Sigma) rabbit antibody and mouse anti-ERα monoclonal antibody (SC-56833) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A + laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
7
Immunofluorescence Imaging of TRIM3 and p53
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MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-TRIM3 polyclonal antibody (HAP043396, Sigma) and mouse anti-P53 monoclonal antibodies (SC-126, Santa Cruz) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
8
Detecting ER Alpha Ubiquitination in Cells
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To directly detect the enriched overall ubiquitinated, K63-ubiquitinated, mono-ubiquitinated and K48-ubiqutinated ER alpha from the cell extracts, HEK293 cells were transfected with 4 ug Ub, 4 ug K63 Ubi, 4 ug Ub-KO or 4 ug K48 Ubi plasmid, 2 ug ER alpha together with 0.5 ug Myc-TRIM3 or Myc-vector. After 48 h, total protein was extracted and pre-cleared with 20ul protein A (santa cruz, SC-2001) for 2 h. The supernatant was collected and immunoprecipitated by ER alpha antibody. Western blot with HA antibody was performed to detect specific ubiquitinated ER alpha. Immunofluorescence assay MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit Anti-TRIM3 (HAP043396, Sigma) and mouse anti-ERα monoclonal antibodies (SC-56833) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
9
Immunofluorescence Imaging of RNF187 and YAP
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EC109 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-RNF187 polyclonal antibody (HAP030098, Sigma, 1/50) and mouse anti-YAP monoclonal antibodies (SC-101199, Santa Cruz, 1/50) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60× oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ.
10
Immunofluorescence Visualization of ZNF213 and ER-α
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MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-ZNF213 (HAP035000, Sigma) and mouse anti-ER alpha monoclonal antibodies (SC-56833) were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA, USA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60× oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using Image J.
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