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Our Abpromise guarantee covers the use of ab13970 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||1/500 - 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
The concentrations of fixative for the IHC applications were typically 10% formalin or 2% paraformaldehyde.
|IHC - Wholemount||Use at an assay dependent concentration.|
|IHC-FrFl||Use at an assay dependent concentration.|
Used at a dilution of 1/2000 for 1 hr (see Abreview for further information).
ab13970 staining GFP in Human U2OS cells by ICC/IF. Cells were paraformaldehyde fixed, permeabilized with 0.5% triton and blocked with 2% antibody dilution buffer for 2 hours. Cells were incubated with the primary antibody (1/1000) for 1 hour at 25°C. An undiluted Alexa Fluor® 488 conjugated Goat anti-chicken polyclonal was used as the secondary antibody.
Image is courtesy of an AbReview submitted by Dr Francois Daubeuf.
Western blot analysis of HEK293 transfected and untransfected cell lysates, labelling GFP with ab13970. Cells were treated by mixing in RIPA buffer and denaturating in Laemmli buffer for 5 mins at 95°C. The gel was Precast at 4-12%. Blocking was with 0.5% milk at 20°C for 5 mins.
ab13970 staining mouse olfactory bulb tissue sections by IHC-Fr. Sections were PFA fixed, permeabilized in 0.4% Triton-X and blocked with 5% serum for 2 hours at 25°C. The primary antibody was diluted 1/1000 and incubated with the sample for 16 hours at 4°C. An Alexa Fluor® 488 conjugated goat anti-chicken was used as the secondary.
Western blot of transgenic mouse spinal cords showing that the rabbit anti-GFP (lane 1) and the chicken anti-GFP (Abcam; lane 2) recognize a band at the same molecular weight.Western blot of transgenic mouse spinal cords showing that the rabbit anti-GFP (lane 1) and the chicken anti-GFP (Abcam; lane 2) recognize a band at the same molecular weight.
Transgenic mice expressing GFP selectively in lamina II of the spinal cord. In the right panels, note the correspondance between the green (rabbit anti-GFP) and red signals (chicken anti-GFP from Abcam) indicating that these two antibody preparations recognized the same gene product. The secondary antibody used with ab13970 was a FITC-labeled goat anti-chicken
ab13970 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 ab13970 at 1/2000 dilution overnight at +4°C followed by incubation with ab150173, Goat polyclonal Secondary Antibody to Chicken IgY - H&L (Alexa Fluor® 488), 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 ab13970 was applied gave a stronger signal in the 488 channel, indicating that ab13970 is binding to GFP and therefore eliciting signal amplification.
ab13970 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).
ab13970 staining mouse olfactory epithelium tissue sections by IHC-Fr. The sample was PFA fixed and blocked with 4% BSA/ 5% NFDM/ 10% NDS for 15 minutes at 20°C. The primary antibody was diluted 1/1000 and incubated with the sample for 1 hour. A FITC conjugated goat anti-chicken was used as the secondary.
This colony is the result of retroviral infection with a control virus. The GFP is under the control of an IRES promoter, so its expression is independent of any other protein. The counter-stain is hoescht.
Image courtesy of an anonymous Abreview.
ab13970 staining GFP + tumor in mouse muscle cells by ICC/IF. Cells were formaldehyde fixed and blocked with 3% BSA for 1 hour at 24°C prior to incubation with the primary antibody (1/500) for 1 hour at 24°C. An Alexa Fluor® 488 conjugated goat anti-chicken was used as the secondary.
ab13970 staining GFP in mouse brain tissue section by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed with paraformaldehyde and permeabilized with 0.1% Triton X100 before blocking with 10% serum for 30 minutes at 250C. The sample was incubated with primary antibody (1/2000) for 16 hours at 250C in 10% NGS in PBS + 0.1% TX100. An Alexa Fluor®488-conjugated Goat polyclonal to chicken IgG was used as secondary antibody at 1/400 dilution. In the image, green staining represents GFP expressed in oligodendrocytes, blue is for ToPro3.
Immunocytochemical immunoflurescence analysis of human cytospined HEK293 cells transfected with GFP, labelling GFP with ab13970 at 1/200 incubated for 16 hours at 4°C with 1% BSA in PBS. Secondary used was a donkey anti-chicken polyclonal DyLight® 594 at 1/500. GFP is shown in red (DyLight® 594). Nuclei are counterstained in blue (DAPI). The left pane shows HEK293 cells transfected with GFP and the right pane shows non-transfected HEK293 cells.