[Histonet] Staining Brain Sections with NeuN

[Sohail Ejaz]

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)