IHC image of beta III Tubulin staining in a section of formalin-fixed paraffin-embedded normal human cerebellum performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab231084, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

All lanes :

Lane 1 : HAP1 whole cell lysate
Lane 2 : HAP1 TUBB3 knockout whole cell lysate
Lane 3 : Neuro2a whole cell lysate
Lane 4 : PC12 whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 50 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

ab231084 was shown to specifically react with beta III Tubulin (TUBB3) in wild type HAP1 cells. No band was observed when beta III Tubulin (TUBB3) knockout samples were used. Wild-type and beta III Tubulin (TUBB3) knockout samples were subjected to SDS-PAGE. ab231084 and ab181602 (Rabbit anti GAPDH) were incubated overnight at 4°C at 1ug/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab231084 staining beta III tubulin (shown in green) in wild-type HAP1 cells (top panel) and TUBB3 knockout HAP1 cells (bottom panel).

The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab231084 at 1 μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

IHC image of beta III Tubulin staining in a section of formalin-fixed paraffin-embedded normal rat cerebellum performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab231084, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.