Immunohistochemistry analysis of GAD2/GAD65 showing staining in the cytoplasm and membrane of paraffin-embedded human pancreatic tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a GAD2/GAD65 mouse monoclonal antibody (398200) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Mouse / IgG1, kappa|
|Immunogen||Recombinant protein corresponding to the N-terminal region of the human GAD65 protein|
|Storage buffer||PBS, pH 7.4|
|Contains||0.1% sodium azide|
|Tested Applications||Dilution *|
|ELISA (ELISA)||Assay Dependent|
|Immunohistochemistry (Paraffin) (IHC (P))||1:10-1:50|
|Western Blot (WB)||1-3 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Antibodies to glutamic acid decarboxylase-65 (GAD65) are present in autoimmune disorders such as insulin-dependent (type1) diabetes mellitus (IDDM), stiff man syndrome and polyendocrine autoimmune disease. Auto-antibodies to GAD65 are present in 60-70% of individuals with newly diagnosed IDDM and thus are important markers for disease activity. These auto-antibodies usually recognize conformation-dependent regions on GAD65 and rarely bind to the second isoform, GAD67. Auto-antibodies to GAD67 are found in only 15% of recent-onset IDDM patients and most of this binding can be blocked with GAD65, suggesting shared epitopes between the two isoforms of GAD.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.