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Data points (n = 30) were collected from three different experiment and showed significant upregulation of GAC and downregulation in KGA upon stimulation under normoxic conditions (****p< 0.0001).
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(***p< 0.0001 **p< 0.005) (E) Mean grey values for cells stained with α-GAC and α-KGA were determined by using the Image J software to determine individual image pixel intensity. This showed that mitochondrial biomass was significantly increased upon stimulation both hypoxia and normoxia. (D) Mitochondrial biomass was determined using Mitotracker Green FM and flow cytometry. The images are representative examples of three different experiments and showed apparent co-localization of GAC and KGA with mitochondria (Bar 10 μm). Cell nuclei were stained using DAPI (blue) and images were merged.
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Localization of KGA, GAC and GLUL relative to mitofilin in quiescent and activated CD4+ T cells.α-CD3/CD28 stimulated (+) and unstimulated (-) CD4+ T cells incubated under hypoxia (H) and normoxia (N) were examined by confocal microscopy after immunofluorescence staining using Alexa Flour 488 (green) conjugated α-KGA (A), α-GAC (B) and α-GLUL (C) and α-mitofilin conjugated with Alexa-Fluor-647 (red). Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license. The results are given as, for example, the percentage of protein A (HIF-2α) that co-localized with protein B (HIF-1β or RNAPII) and vice versa. method to automatically create a threshold prior to calculating the Mander's coefficient for both proteins. (b) Immunofluorescence images were analysed using ImageJ plugin Co-localization Threshold with use of the Costes et al. Abbreviations: RNAPII, RNA Polymerase II phospho-serine 5.
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White arrows highlight regions of co-localization. Inlay is the magnified region (white square). ‘Overlay’ is the co-localization image superimposed onto the merged image. White indicates pixels where both red and green signal is found (i.e.
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‘Coloc’ is the co-localization channel calculated using ImageJ plugin Co-localization Threshold. ‘Merge’ is the red image superimposed onto the green image of the co-stained nuclear proteins. (a) Immunofluorescence detecting indicated proteins using antibodies labelled with Alexa Fluor 555 (red, pseudocolour) and Alexa Fluor 488 (green, pseudocolour). Image collected and cropped by CiteAb from the following publication (), licensed under a CC BY license.Ĭo-localization of HIF-2α with HIF-1β and RNAPII. (b) Immunofluorescent images (a) were analysed using ImageJ plugin Co-localization Threshold as in figure 3. Abbreviations: PML, Promyelocytic leukemia protein HDAC5, histone deacetylase 5 HAF, hypoxia associated factor YFP, yellow fluorescent protein. ‘Overlay’ is the merge image with the colocalization image superimposed. ‘Merge’ is HIF-2α (red) image superimposed onto the green image of the co-detected nuclear protein. Other nuclear proteins (SC35 and HAF) were detected with a secondary antibody coupled to Alexa Fluor 488 (green, pseudocolour) or were fused to YFP for direct detection (PML and HDAC5). Endogenous HIF-2α is labelled with a secondary antibody coupled to Alexa Fluor 555 (red, pseudocolour). (a) Immunofluorescent images of HIF-2α and other nuclear proteins that are known to localize into nuclear bodies. The images were captured at 60X magnification.Ĭo-localization HIF-2α and other nuclear proteins. No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e). F-actin was stained with Rhodamine Phalloidin (Product # R415, 1:300) (Panel c: red). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938). Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa Fluor® 488 conjugate (Product # A-11008) was used at a concentration of 4 µg/mL in phosphate buffered saline containing 0.2% BSA for 45 minutes at room temperature, for detection of alpha Tubulin in the cytoplasm (Panel a: green). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 2 µg/mL Rabbit primary antibody for 3 hours at room temperature. Immunofluorescence analysis of Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa Fluor® 488 conjugate was performed using HeLa cells stained with alpha Tubulin Rabbit Polyclonal Antibody (Product # PA5-16891).