Recombinant Anti-GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) antibody [EPR933Y] (ab68476)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue labelling GSK3 (alpha + beta) with purified ab68476 at 1/2000. Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. ab209101, a Rabbit specific IHC polymer detection kit HRP/DAB was used. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Positive staining on human thyroid carcinoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B).

The section was incubated with ab68476 for 30 minutes at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

Blocking and diluting buffer: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling GSK3 (alpha + beta) with purified ab68476 at 1/2000. Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. ab209101, a Rabbit specific IHC polymer detection kit HRP/DAB was used. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Positive staining on human ovarian carcinoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B).

The section was incubated with ab68476 for 30 minutes at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue sections labeling GSK3 (alpha + beta) with Purified ab68476 at 1:50 dilution (5.3 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Blocking and diluting buffer: 5% NFDM/TBST

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Human brain tissue stained with unpurified ab68476 at 1/100 dilution.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

GSK3 (alpha + beta) (phospho Y216 + Y279) was immunoprecipitated from 10μg HeLa (human cervix adenocarcinoma) whole cell lysate with ab68476 at 1/50 dilution (2μg in 0.35mg lysates). Western blot was performed from the immunoprecipitate using ab68476 at 1/200 dilution (9 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 (Input): HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate 10μg
Lane 2 (+): HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab68476 in HeLa (human cervix adenocarcinoma) treated with 1uM staurosporine for 4h whole cell lysate

Exposure Time: 30 seconds
Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Blocking and dilution buffer: 5% NFDM/TBST

Dot Blot analysis of Lane 1: Human GSK3 (alpha + beta) (pY216 + pY279) phospho peptide and Lane 2: Human GSK3 (alpha + beta) non-phospho peptide labeling GSK3 (alpha + beta) (phospho Y216 + Y279) with ab68476 at 1/1000 dilution. 5% NFDM/TBST was used as the diluting and blocking buffer. ab97051 Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100000 dilution. Exposure time: 3 minutes.