Anti-Phosphotyrosine antibody [PY20] (ab10321)

Immunohistochemistry - Free Floating - Phosphotyrosine antibody [PY20] (ab10321)

Immunofluorescent staining with ab10321 mouse monoclonal [PY20] phosphotyrosine antibody in the rat cortex. Cells stained appear to be microglial cells. Picture taken with objective X20. Protocol: IHC free-floating protocol using 4%PFA fixed brain tissue. Rats were intracardially perfused with 4% PFA. Tissue was post-fixed overnight in the same fixative, cryoprotected in 20% sucrose and frozen in OCT. Primary antibody ab10321 was used at 1ug/ml incubated overnight at room temperature. Secondary antibody was Alexa Fluor 488 used at 1/1000, 2h incubation at room temperature. Image recoloured in Adobe photoshop.

Sophie Pezet, Univ London Kings Coll, United Kingdom

Immunohistochemistry - Free Floating - Phosphotyrosine antibody [PY20] (ab10321)

Immunofluorescent staining with ab10321 mouse monoclonal [PY20] phosphotyrosine antibody in the rat spinal cord. Cells stained appear to be microglial cells. Picture taken with  X40 objective. Protocol: IHC free-floating protocol using 4% PFA fixed spinal cord tissue. Rats were intracardially perfused with 4% PFA. Tissue was post-fixed overnight in the same fixative, cryoprotected in 20% sucrose and frozen in OCT. Primary antibody ab10321 was used at 1ug/ml incubated overnight at room temperature. Secondary antibody was Alexa Fluor 488 used at 1/1000, 2h incubation at room temperature. Image recoloured in Adobe photoshop.

Sophie Pezet, Univ London Kings Coll, United Kingdom

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Phosphotyrosine antibody [PY20] (ab10321)

IHC image of ab10321 staining in Human Normal Hippocampus formalin fixed paraffin embedded tissue section, 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 20 mins. The section was then incubated with ab10321, 1µg/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.

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.

Western blot - Phosphotyrosine antibody [PY20] (ab10321)

All lanes : Anti-Phosphotyrosine antibody [PY20] (ab10321) at 1 µg/ml

Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 2 : NIH 3T3 treated with Vanadate and PDGF Whole Cell Lysate

Lysates/proteins at 5 µg per lane.

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
developed using the ECL technique

Performed under reducing conditions.

Exposure time : 1 minute

Cells were serum starved overnight and then incubated at room temperature for 10mins in a final concentration of 1mM sodium vanadate. PDGF was then added at a final concentration of 5ng/ml and cells were incubated at 37ºC for 30mins. Vanadate inhibits endogenous phosphatases and PDGF stimulates phosphorylation. Western blots of NIH 3T3 cell lysates treated with vanadate and PDGF show an array of phosphorylated tyrosine compared to controls.