Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100-permeabilised mouse cerebrum tissue staining glutamine synthetase with ab176562 at 1/250 dilution, followed by alexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). DAPI was used as a nuclear counterstain.

Positive staining on mouse cerebrum (PMID: 23895693).

All lanes : Anti-Glutamine Synthetase antibody [EPR13022(B)] (ab176562) at 1 µg

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : GLUL knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 42 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab176562 observed at 42 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab176562 was shown to recognize Glutamine Synthetase in wild-type HAP1 cells as signal was lost at the expected MW in GLUL knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GLUL knockout samples were subjected to SDS-PAGE. Ab176562 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded, Human glioma tissue labeling Glutamine Synthetase with unpurified ab176562 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-Glutamine Synthetase antibody [EPR13022(B)] (ab176562) at 1/1000 dilution (unpurified)

Lane 1 : Human fetal liver lysate
Lane 2 : Jurkat cell lysate
Lane 3 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Developed using the ECL technique.

Predicted band size: 42 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human glioma tissue sections labeling Glutamine Synthetase with purified ab176562 at 1:500 dilution (0.18 μg/ml). Heat mediated antigen retrieval was performed using citrate Buffer, pH6.0. 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.

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100-permeabilised rat cerebrum tissue staining glutamine synthetase with ab176562 at 1/250 dilution, followed by alexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. Heat mediated antigen retrieval was performed using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). DAPI was used as a nuclear counterstain.

Positive staining on rat cerebrum (PMID: 23895693).

Immunohistochemical analysis of formalin-fixed, paraffin-embedded, Human liver tissue labeling Glutamine Synthetase with unpurified ab176562 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-Glutamine Synthetase antibody [EPR13022(B)] (ab176562) at 1/2000 dilution (purified)

Lane 1 : Human fetal liver lysates at 20 µg
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg
Lane 3 : Mouse spleen lysates at 20 µg
Lane 4 : Rat spleen lysates at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa

Blocking and diluting buffer: 5% NFDM/TBST

ab176562 staining Glutamine Synthetasein Mouse Liver tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with formaldehyde, blocked with PB ab64226 for 10 minutes at room temperature and antigen retrieval was by heat mediation in citrate buffer. The sample was incubated with primary antibody (1/200) for 30 minutes. A HRP-conjugated Goat anti-rabbit polyclonal (1/200) was used as the secondary antibody.

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