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For each field, three images were captured: DAPI (nuclei, blue), GFP (AR, green), and dsRED2skl (reporter protein, red).
We thank the Ministry of Higher Education (Egypt) for funding MA Aleskandarany, and the Breast Cancer Campaign for funding AR Green.
AlexaFluor 488 (Life Technologies) conjugated goat anti-rabbit secondary antibody was used to detect AR (green channel), and AlexaFluor 594 conjugated goat anti-mouse secondary antibody (Life Technologies) was used for Jarid1b-myc-his (red channel).
We acknowledge the support of the University of Nottingham and Nottingham University Hospitals Trust in funding DG Powe, G Akhtar, EA Rakha and IOE, We thank the Ministry of Higher Education (Egypt) for funding HO Habashy, TM Abdel-Fatah and E Rakha, and Breast Cancer Campaign for funding AR Green.
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For each cell line, 100 interphase nuclei were scored and the ratio of red (AR) to green (Xcen) signals was calculated.
Representative z-stack images of NSC-34 cells that were transfected as indicated, treated with 10 nM DHT, and subjected to immunofluorescence using anti-AR N20 (green; A – C ) and anti-FLAG (red; A′ – C′ ) antibodies to detect AR and NLK expression, respectively.
Representative z-stack images of NSC-34 cells that were transfected as indicated, treated with ethanol (as a negative control for DHT), and subjected to immunofluorescence using anti-AR N20 (green; A – C ) and anti-FLAG (red; A′ – C′ ) antibodies to detect AR and NLK expression, respectively.
Immunofluorescent staining was acquired by an Olympus BX61 microscope equipped with an Olympus confocal scan unit FV500 equipped with two laser lines, Ar-Kr green (488 nm) and He-Ne red (646 nm) used to detect the NC marker, 5-CFDA, and secondary antibodies conjugated at Alexa 555, respectively.
The analysis was done in HeLa cell lines stably transfected with wild type or mutant ARs fused with green fluorescent protein, or using AR immunofluorescence in primary cultures of genital skin fibroblasts generated from foreskin biopsies of historical AIS patients, or of normal neonates undergoing circumcision at birth.
3. Bradley AR, Wall ID, Green DV, Deane CM, Marsden BD.
NSC-34 cells were treated with DHT as indicated and subjected to immunofluorescence using anti-AR N-20 (green) and anti-FLAG (red) antibodies to detect AR aggregation and NLK co-expression, respectively.
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