[Histonet] Staining Brain Sections with NeuN

[Harlem Kaputnik]

What did your control tissue tell you.... as in staining of the positive control, and your negative positive control as well?
 
> Date: Sat, 20 Feb 2010 10:14:22 -0800
> From: sohail_e <@t> yahoo.com
> To: histonet <@t> lists.utsouthwestern.edu
> CC: histonet-request <@t> lists.utsouthwestern.edu; histonet-owner <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Staining Brain Sections with NeuN
> 
> 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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