[Histonet] Re: Troubleshooting IHC

[Adam .]

Yes. I block in 3% H2O2, followed by protein block (it's a mysterious buffer
called TNB that comes with the tyramide amplification kit), and then
avidin/biotin.

Adam

On Thu, Mar 25, 2010 at 2:25 PM, Margaryan, Naira <
NMargaryan <@t> childrensmemorial.org> wrote:

>  Hi Adam,
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> How do you block?
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> I usually have: H2O2, Avidin/Biotin and Protein blocking steps.
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> Naira
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> Message: 12
>
> Date: Thu, 25 Mar 2010 10:43:28 -0500
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> From: "Adam ." <anonwums1 <@t> gmail.com>
>
> Subject: [Histonet] Troubleshooting IHC
>
> To: histonet <@t> lists.utsouthwestern.edu
>
> Message-ID:
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>       <858249121003250843h2d0f16a2q1a74ad1091630300 <@t> mail.gmail.com>
>
> Content-Type: text/plain; charset=ISO-8859-1
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>
>
> Hi all,
>
>
>
> I've recently run into a problem troubleshooting IHC on mouse bones. I am
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> using the tyramide amplification system using a goat primary, anti-goat
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> biotin, strepavidin-HRP, biotinyl tyramide, followed by another
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> strepavidin-HRP and then DAB+ from Dako. When I use an isotype goat IgG, I
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> get essentially no staining.
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> I titered it once for immunofluorescence (SA-fluor instead of HRP in the
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> last step) and got a titer of 1:800, and these staining conditions are
> quite
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> reproducible. Then I titered for IHC, and 1:200 gave the best signal to
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> noise. I did a batch of staining using those IHC conditions, and it stained
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> beautifully, comparable to my IF. I then tried to repeat it on a second
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> batch of sections processed in the same way, and the background was
> terrible
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> (but not on my isotype slide). I thought maybe it was a problem in
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> processing so I did a third batch of sections, and the background was still
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> really bad. I see real staining sometimes, but I need to quantify this
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> staining using histomorphometry, so I really need clean staining. Any
> ideas?
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> The only thing I can think of is that 1:200 is just at the limit of
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> titration that gives too much background.
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> Thanks,
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> Adam
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