Dear Friends I am staining brain sections (30 micro meter) with NeuN antibody to evaluate neuronal loss after stroke in Rat.
I am using under mentioned protocol which worked fine during first attempt but afterward i am not getting any signal. Please have a look at the protocol and guide me where i am wrong. Quench the free floating sections (10% methanol and 10% H2O2 in distilled water) for 5 min. Wash the sections in Trizma Buffer Saline (TBS) (pH7.4)…….3 X 5 min. Block the sections in 3% normal horse serum with TBS containing 0.2% Triton X-100 (TXTBS) for 1 h at room temperature (RT). Incubate the sections overnight at (RT) with primary antibody (NeuN, monoclonal: dilution 1:1000; MAB377, Chemicon International) in TXTBS containing 1% NHS. Wash the sections with TBS …….3 X 5 min. Incubate the sections for 3 h (RT) with biotinylated anti-mouse IgG (rat- absorbed: dilution 1:200; Vector) secondary antibody in TXTBS with 1% NHS Wash with TBS …….3 X 5 min. Incubate the sections with a streptavidin-biotinylated horseradish peroxidase complex (VECTASTAIN Elite ABC Kit) for 2 h (RT).Wash the sections in TBS …….3 X 5 min. Wash the sections with PBS Incubate the sections with DAB Peroxidase substrate kit. Remove excess stain by thorough washing in PBS Mount the sections onto 1% gelatin-coated slides and air-dry overnight (RT) _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet