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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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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Anti-GFP antibody (ab290) at 1/2500 dilution + Recombinant A. victoria GFP protein (ab84191) at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Observed band size: 27 kDa

why is the actual band size different from the predicted?


Exposure time: 30 seconds

Secondary antibody - goat anti-rabbit HRP preadsorbed (ab97080)

All lanes : Anti-GFP antibody (ab290) at 1/5000 dilution

Lane 1 : LNCaP whole cell lysate - pEGFP empty vector
Lane 2 : LNCaP whole cell lysate - pEGFP-PKD1 transfected

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Exposure time: 10 seconds

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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All lanes : Anti-GFP antibody (ab290) at 1/5000 dilution

Lane 1 : COS7 whole cell lysate - transfected with GFP-Eml4
Lane 2 : COS7 whole cell lysate - transfected with GFP

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP-conjugated pig anti-rabbit IgG at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Observed band size: 30 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

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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