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 < nmargar...@childrensmemorial.org> wrote: > Hi Adam, > > > > How do you block? > > > > I usually have: H2O2, Avidin/Biotin and Protein blocking steps. > > > > Naira > > > > Message: 12 > > Date: Thu, 25 Mar 2010 10:43:28 -0500 > > From: "Adam ." <anonwu...@gmail.com> > > Subject: [Histonet] Troubleshooting IHC > > To: histonet@lists.utsouthwestern.edu > > Message-ID: > > <858249121003250843h2d0f16a2q1a74ad1091630...@mail.gmail.com> > > Content-Type: text/plain; charset=ISO-8859-1 > > > > Hi all, > > > > I've recently run into a problem troubleshooting IHC on mouse bones. I am > > using the tyramide amplification system using a goat primary, anti-goat > > biotin, strepavidin-HRP, biotinyl tyramide, followed by another > > strepavidin-HRP and then DAB+ from Dako. When I use an isotype goat IgG, I > > get essentially no staining. > > > > I titered it once for immunofluorescence (SA-fluor instead of HRP in the > > last step) and got a titer of 1:800, and these staining conditions are > quite > > reproducible. Then I titered for IHC, and 1:200 gave the best signal to > > noise. I did a batch of staining using those IHC conditions, and it stained > > beautifully, comparable to my IF. I then tried to repeat it on a second > > batch of sections processed in the same way, and the background was > terrible > > (but not on my isotype slide). I thought maybe it was a problem in > > processing so I did a third batch of sections, and the background was still > > really bad. I see real staining sometimes, but I need to quantify this > > staining using histomorphometry, so I really need clean staining. Any > ideas? > > The only thing I can think of is that 1:200 is just at the limit of > > titration that gives too much background. > > > > Thanks, > > Adam > > > _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet