[Histonet] Staining Brain Sections with NeuN
Sohail Ejaz
sohail_e <@t> yahoo.com
Sat Feb 20 12:14:22 CST 2010
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)
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