Characterisation of HDAC4-trapped pGAL4-driven expression using anti-gfp antibody ab290.

A. Molecular organisation of the HDAC4 gene locus. The 22.6 kb locus is shown, with exons depicted as boxes. Translated regions are black boxes. The P element transposons NP3558 and NP1617 are inserted 3144 and 4838 bp downstream from the 3' end of the 2nd exon, respectively. The site of YFP insertion into HDAC4:YFP at 1310 bp downstream from the 3' end of the 2nd exon is also shown. YFP is flanked by splice sites, which facilitate internal incorporation of YFP into the HDAC4 protein. B-D. Confocal microscopy images of whole mount brains with NP1617-driven expression of nuclear localized dsRED and CD8::GFP.

B. Frontal confocal projection through the brain showing CD8::GFP in the mushroom body and nuclear localized dsRED in the Kenyon cell nuclei. Some expression can be seen at lower levels in other regions of the brain. Scale bar = 200 μm for B,C,E,F.

C. Posterior view showing CD8::GFP localized to the calyx and nuclear localized dsRED in the Kenyon cell nuclei.

D. CD8::GFP expression is observed in α/β and γ lobes of the MB. Immunohistochemistry against Trio (blue) which is expressed in the α'/β' and γ lobes of the mushroom body, confirms co-localization of GFP with Trio in the γ but not the α'/β' lobes. Scale bar = 50 μm for D and G.

E-G. Confocal microscopy images of whole mount brains with NP3558-driven expression of CD8::GFP. E. Frontal confocal projection showing CD8::GFP in the mushroom body and widespread through the rest of the brain. F. Posterior view showing high expression of GFP in the Kenyon cells. G. Anti-Trio immunohistochemistry reveals colocalization of GFP and Trio in the α'/β' and γ lobes, and GFP is also observed in the Trio-negative α/β lobes.

Bone marrow-derived infiltrating cells in the stromal tissue of gastric intraepithelial tumor traced by GFP direct fluorescence.

(A) Normal tissues of the glandular stomach of a regular GFP(−) control mouse. (B) Normal tissues of the glandular stomach of a GFP(+) transgenic control mouse; (C, E, D, F) An induced gastric intraepithelial neoplasia (GIN) in a bone marrow transplanted mouse. GFP(+) BMDCs tracked with direct fluorescence localized in the GIN stromal tissue are shown in C and E. The same GIN lesion slide stained by H&E after the fluorescence observation are shown in D and F. DAPI (A–C and E) and hematoxylin (D and F) are used to visualize nuclei, respectively. Locations of the images C and D in the images E and F, and the image E in the image F are marked in the corresponding color. The gastric glands and stromal cells are also labeled.

Immunohistochemistry (Free Floating) analysis of mouse brain tissue sections labelling GFP with ab290. Tissue was fixed with 4% PFA, frozen 30 µm sections were blocked for 1 hour at room temperature with 10% normal goat serum + donkey anti-mouse IgG Fab fragments (0.1 mg/ml). Sections were incubated with the primary antibody at a dilution of 1/1000 in TBS + 0.25% Triton-X for 16 hours at 4°C. A Cy2^®-conjugated donkey anti-rabbit IgG (H+L) at a dilution of 1/200 was used as the secondary antibody.

Image shows anti-NeuN (red), DAPI (blue), and anti-GFP staining of GFP-cre (green, yellow with NeuN colocalization).

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ab290 staining dog hearts (Adv-GFP injection) tissue sections by IHC-P.  Sections were PFA fixed and subjected to heat mediated antigen retrieval in citric acid (Ph6.0, 0.05% Tween20) prior to blocking with 10% serum for 30 mins at 37°C.  The primary antibody was diluted 1/1000 in PBS and incubated with the sample for 1 hour at 25°C.  A HRP conjugated secondary like  Goat Anti-Rabbit IgG H&L (HRP) (ab205718) was used.

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Immunofluorescence images showing similar localization of Yes-GFP (first 10 aa's of Yes PTK fused to the N-terminus of GFP) to full length Yes PTK. A: Distribution of Yes detected using mouse anti-Yes Ab followed by Texas Red-conjugated anti-mouse Ab. B: Chimeric GFP's detected using rabbit anti-GFP Ab (Abcam ab290) followed by FITC-conjugated anti-rabbit Ab.

Image kindly provided by L.G. Berthiaume. Taken from J. McCabe and L.G. Berthiaume, Functional Roles for Fatty Acylated Amino-terminal Domains in Subcellular Localization, Molecular Biology of the Cell 10:3771-3786, 1999

ab290 immunoprecipitating GFP in HEK293 nuclear lysate expressing GFP. 20µg of lysate was incubated with primary antibody (1 µg/mg lysate) and matrix (Protein G) for 16 hours at 4°C in AFC low salt buffer. For western blotting ab290 (1/5000) was used to confirm successful immunoprecipation.

Lane 1: HEK293 nuclear lysate expressing GFP input.
Lane 2: IP of HEK293 nuclear lysate expressing GFP.
Lane 3: Cells with no GFP.

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ab290 staining GFP in GFP-transfected NIH3T3 cells. The cells were fixed with 4% formaldehyde (10min) and then blocked in 1% BSA / 0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab290 at 1/200 dilution overnight at +4°C followed by incubation with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081), for 1 hour, at 1μg/ml.

Under identical experimental conditions, when compared to the basal level of GFP expression in transfected NIH3T3 cells, the cells upon which ab290 was applied gave a stronger signal in the 488 channel, indicating that ab290 is binding to GFP and therefore eliciting signal amplification.

ab290 was also applied to non-GFP-transfected NIH3T3 cells, which produced no positive staining, indicating specificity for GFP. Nuclear DNA was labelled with 1.43μM DAPI (blue).

ab290 immunoprecipitate in human HEK293 cells transfected with Annexin1-GFP. 25µg of cell lysate was incubated with the primary antibody and matrix (Protein G) in 1% TX-100, 10% glycerol, 1X PBS for 16 hours at 4°C. For Western blotting anti-rabbit HRP conjugated secondary antibody was used at a dilution at 1/5000.

Lane 1: Lysate of HEK293 cells expressing Annexin1-GFP fusion protein.
Lane 2: IP with anti-GFP.
Lane 3: Not bound fraction.

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Flow cytometry analysis of paraformaldehyde-fixed human differentiated hNSCs cells prepared through accutase, quantifying GFP with ab290 diluted 1/300 incubated for 20 hours at 25°C. Permeableization was through 0.25% Triton X-100 in DPBS. Secondary was anti-rabbit Alexa Fluor® 488 conjugated secondary antibody was used  at 1/500. The gating strategy was against undifferentiated stem cells (shown in white).

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ab290 staining GFP in U2OS cells expressing TRF2-GFP fusion protein by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with NP40 and blocked with 3% BSA for 1 hour at 21°C. Samples were incubated with the primary antibody (1/1000 in PBS + 3% BSA) for 12 hours at 4°C. An Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at a dilution of 1/500 was used as the secondary antibody.

Green - GFP.
Blue - DAPI.

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Blocked with 5% milk for 1 hour at 23°C.

Incubated with the primary antibody for 16 hours at 4°C.

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Blocked with 5% milk for 1 hour at 20°C.

Incubated with the primary antibody for 18 hours at 4°C in TBS containing 2% milk and 1% Tween.

Predicted MW of Eml4 ~ 120 kDa.

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