Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFP antibody [EPR14104] (ab183734)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of GFP transgenic mouse pancreas tissue labelling GFP with purified ab183734 at a dilution of 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunocytochemistry/ Immunofluorescence - Anti-GFP antibody [EPR14104] (ab183734)

ab183734 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 ab183734 at 1/500 dilution overnight at +4°C followed by incubation with ab150081, Goat Anti-Rabbit IgG 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 ab183734 was applied gave a stronger signal in the 488 channel, indicating that ab183734 is binding to GFP and therefore eliciting signal amplification.

ab183734 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).

Western blot - Anti-GFP antibody [EPR14104] (ab183734)

Blocking and dilution buffer: 5% NFDM /TBST.

All lanes:

Western blot - Anti-GFP antibody [EPR14104] (ab183734) at 1/10000 dilution

Lane 1:

GFP transfected 293 cell lysate at 10 µg

Lane 2:

Non-transfected 293 cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 27 kDa

Observed band size: 25 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFP antibody [EPR14104] (ab183734)

Immunohistochemical analysis of paraffin-embedded GFP transgenic mouse colon tissue (left) and normal mouse colon tissue (right) labeling GFP with unpurified ab183734 at 1/250 dilution followed by prediluted HRP Polymer for Rabbit IgG. Counterstained with Hematoxylin.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunocytochemistry/ Immunofluorescence - Anti-GFP antibody [EPR14104] (ab183734)

Immunofluorescent analysis of 4% paraformaldehyde-fixed GFP transfected 293 cells labeling GFP with unpurified ab183734 at 1/500 dilution, followed by Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody at 1/200 dilution (green). Counterstained with Dapi (blue)

Western blot - Anti-GFP antibody [EPR14104] (ab183734)

Different batches of ab183734 were tested on GFP transfected HEK-293 (Human embryonic kidney epithelial cell) lysate at 0.004 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 27 kDa.

All lanes:

Western blot - Anti-GFP antibody [EPR14104] (ab183734)

Predicted band size: 27 kDa