RE: [Histonet] BCIP/NBT with blocking buffer
The way I read your protocol, what you are detecting here is the goat serum
with the rat antigoat, hence you have terrible background. In otherwords,
you have an incorrect secondary antibody. If your primary is made in Rat
host (rat anti-mouse) then your secondary should be goat antiRat, adsorbed
to mouse, or even Donkey antiRat (Jackson).
Make sure your secondary is detecting host of primary, i.e. Rat host or rat
antiMouse needs either goat - or donkey antiRat adsorbed to mouse.
Your normal serum block should match the host of the secondary, and use 5
Do avidin biotin block before the primary, after normal serum
block. Vector has kit, whenever you work with ABC or Strepavidin-AP method.
It is strongly advisable to do a dilution panel on all your primaries
starting with a target concentration of 10 ug/ml and then do a serial
dilution to determine best working concentration of primary. Your
secondaries should be around 2 - 5ug/ml, most of the time that means approx
1:250 dilution. Dilute your secondary with normal serum matched to host of
that secondary, or just use the normal serum block. Donkey antiRat would
diluted in 5% donkey serum, goat antiRat would be diluted in 5% goat serum.
Handling frozen sections, cut and air dry overnight, then fix in 4C acetone
10 min, air dry for 20 min.
At 12:58 PM 12/22/2004, you wrote:
>Ok.....here is my basic procedure. Possibly you would have some suggestions
>to help me troubleshoot. I am working with 3 different primaries. All 3
>have the same background issue. The stain is working though!
>First of all I am using frozen sections 10um thick-mouse embryos
>I fix in 4 degree acetone, air dry, hydrate in PBS
>Block in 1.5% normal goat serum made in 1X PBS
>Primary is made in rat anti-mouse (1:200 in blocking buffer)
>Secondary is rat anti-goat (1:1500 in blocking buffer)
>I'm using Vector's ABC kit
>Rinsing in TBST
>Rinsing in NTMT with 50uL levamisole
>Staining with BCIP/NBT (4.5uL NBT+3.5uL BCIP+1mL NTMT/levamisole
>Rinsing in TBST
>I've also tried this with Vector Red and still had a lot of background. My
>control is no primary-I substitute just blocking buffer and it has as much
>background as the other slides.
>I really appreciate any thoughts you may have. I am on que to restain using
>a 1:400 primary concentration and blocking for 1 hour instead of 30 minutes.
>Oh, I am staining for megakaryocytes.
>Rachael L. Emerson
>Center for Human Genetics and Molecular Pediatric Diseases
>University of Rochester Medical Center
>575 Elmwood Avenue MRBX 1.11301
>Rochester, NY 14642
>Tel (585) 275-5073
>Fax (585) 276-0232
> > ----------
> > From: Gayle Callis
> > Sent: Wednesday, December 22, 2004 2:42 PM
> > To: Emerson, Rachael; Histonet@lists.utsouthwestern.edu
> > Subject: Re: [Histonet] BCIP/NBT with blocking buffer
> > The answer is theroretcially - yes. However, this is not the endogenous
> > enzyme you need to block for this chromogen. NBT/BCIP is a chromogen for
> > alkaline phosphatase method, so blocking endogenous peroxidase is
> > unnecessary. You need to put levamisole in with chromogen to block
> > endogenous alkaline phosphatase found in tissues. Instruction sheets
> > usually tell you this. Levamisole is sold separately i.e. Vector has it.
> > OR are you doing a double stain? with both HRP and AP methods. If that
> > is the case, you would still need to block for endog perox for the the HRP
> > portion of staining AND block with levamisole with AP portion.
> > At 12:18 PM 12/22/2004, you wrote:
> > >Hello. Can anyone tell me if I can block endogenous peroxidase with 0.3%
> > >H2O2 in sodium azide and still visualize with BCIP/NBT on frozen
> > sections?
> > >
> > >Thanks
> > >
> > >
> > >
> > >_______________________________________________
> > >Histonet mailing list
> > >Histonet@lists.utsouthwestern.edu
> > >http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> > Gayle Callis
> > MT,HT,HTL(ASCP)
> > Research Histopathology Supervisor
> > Veterinary Molecular Biology
> > Montana State University - Bozeman
> > PO Box 173610
> > Bozeman MT 59717-3610
> > 406 994-6367 (lab with voice mail)
> > 406 994-4303 (FAX)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)
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