All lanes : Anti-Glutamate Receptor 1 (AMPA subtype) antibody (ab31232) at 1 µg/ml

Lane 1 : Brain (Mouse) Tissue Lysate
Lane 2 : Brain (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 102 kDa
Observed band size: 102 kDa
Additional bands at: 47 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab31232 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Immunohistochemistical detection of Glutamate Receptor 1 (AMPA subtype) with ab31232 on  formaldehyde-fixed paraffin-embedded mouse brain sections. Antigen retrieval step: heat mediated in Citric acid pH6 buffer.Blocking step: 1%BSA for 10 mins @ rt°C. Primary antibody ab31232 incubated at 1/100 for 2 hours. Secondary antibody: anti-rabbit IgGs conjugated to biotin (1/200). The composite image shows immunostaining in cell bodies and processes of a subset of neurones in the mouse cortex (upper image) and also in the dendritic fields of the molecular layer of the mouse cerebellum (lower image). Purkinje cells of the cerebellum are negative but their primary processes are clearly delineated by a punctate membrane-region positivity. What appears to be small cells in close contact with the Purkinje cell bodies are strongly positive (perhaps these are basket cells?).

IHC image of Glutamate Receptor 1 (AMPA subtype) staining in a section of formalin-fixed paraffin-embedded normal rat brain 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 ab31232, 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.

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.

Immunohistochemistical detection of Glutamate Receptor 1 (AMPA subtype) with ab31232 on formaldehyde-fixed paraffin-embedded rat cerebellum sections. Antigen retrieval step: heat mediated  in citric acid pH6. Blocking step: 1% BSA  for 10 mins @ rt°C.  Primary antibody was incubated at 1/200 for 2 hours @ room temp. Secondary antibody: anti-rabbit IgGs conjugated to biotin (1/200). The composite image shows localisation of this protein apparently exclusive to the molecular layer of the cerebellum upper image (low power). In the lower image (higher power), the molecular layer positivity appears to be compartmentalised/restricted.

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