5-HT (Serotonin) 2A Receptor Antibody

We have been interested in detecting changes of 5-HT2a in rodent brain, primarily by immunoblotting, also by immunohistochemistry. We have tested several commercial antibodies, with only your Ab showing a specific signal, as verified by brain distribution, preadsorbtion and by a lack of signal in KO animals. We have optimized a immunoblotting protocol which works well to detect 5-HT2a in rat brain lysates. Mouse tissue is a little bit more difficult but still possible. Immunoblotting: Rat or mouse brain samples were lysed by brief sonication in a buffer containing: 1% SDS in 1xPBS, protease inhibitors (Complete Mini, Roche). Samples were mixed with a modified Leamli buffer (containing a reducing agent; 40mM DTT), incubated at 37C for 30mins and separated by SDS-PAGE. Separated proteins were then blotted onto a PVDF membrane, blocked with 5% milk/TBS/0.1% Tween80 and probed with anti-5HT2a antibody (1:500, Immunostar #24288) followed by anti-rabbit HRP- conjugated secondary antibody (1:5000, Jackson Immuno). Signal was visualized using ECL2 kit according to manufacturer's instructions (Thermo). Image Samples: 1 (Rat prefrontal cortex, 25ug), 2 (Rat laterodorsal thalamus, 25ug), 3 (Wt mouse cortex, 25 ug), 4 (5-HT2a KO mouse cortex, 25 ug), 5 (Wt mouse cortex, 75 ug), 6 (5-HT2a KO mouse cortex, 75 ug). As expected, sample in lane 2 showed low expression, and samples in lanes 4 and 6 showed no expression of 5-HT2a receptors. Mouse tissues obtained from Dr. Gonzalez-Maeso (Mount Sinai Hospital, NY) indicates detected 5Ht2a receptor signal.

Here are the results of staining of the mouse 5-HT 2A receptors in the brain with the use of Immunostar's antibodies (Cat no: 24288). I think the antibodies are specific for the mouse receptor (no receptor labeling in the brain of 5-HT 2A receptor knockout mouse) and can be used in a Western blot. What I already found out is that 5-HT 2A receptors in the cortex seem to be glycosylated (band around 75 kDa) since incubation with PNGaseF releases the protein that is around 50 kDa. We received the 5-HT 2A receptor knockout animals from another research team in Paris.