[Histonet] IHC theoretical question

[Adam .]

Hi,

I find this confusing myself sometimes, so I'll try my best to explain this.
I stain mouse tissues. Let's say that I want to stain for the classic
macrophage F4/80 marker, so I go buy a rat anti-mouse F4/80. In order to
further amplify, I buy a biotinylated donkey anti-rat antibody, and then
detect that with HRP/DAB.

Certain parts of the section may be sticky and nonspecifically bind proteins
or even have an affinity for antibodies. If I didn't block, my primary and
secondary antibodies might just bind to them, adding a lot of background.
Pre-incubating with protein such as serum, BSA, or milk will bind to those
and prevent nonspecific binding of your primary or secondary antibodies.
Serum is typically used because it contains antibodies, so you would also
saturate anything that is nonspecifically but preferentially binding
antibodies such as Fc receptors (although sometimes you have add Fc block if
this is particularly problematic).

Typically serum from the secondary antibody host (in this case, donkey) is
used. Let's say your tissue has sticky parts and instead of blocking with
donkey serum as you should, you accidentally block with rat serum. Now your
entire section is coated with low levels of rat IgG that was in your rat
serum, so when you add your anti-rat secondary antibody, it will bind
everywhere. If you block with rabbit serum, there still is a chance that
your anti-rat secondary may cross react with the rabbit IgG. Now if you use
donkey serum, there is pretty much no chance that the donkey raised
antibodies against donkey IgG unless the donkey had some bizarre autoimmune
disease. So that's why you typically add serum derived from the secondary
antibody host.

Hopefully, your antigen doesn't nonspecifically bind stuff, or else IHC is
going to be very hard. Most antigens don't nonspecifically bind stuff, so
you're good.

Is that clear?
Adam

On Fri, Dec 24, 2010 at 10:30 AM, wassan alkadhumi <w_alkadhumi <@t> yahoo.com>wrote:

> Dear histonet members
> I have a theoretical question concerning IHC, we do HRP method using DAKO
> materials, first step in immuon staining is to add peroxidase blocking
> solution
> to quench endogenus peroxidase. The second step is what am having problem
> with,
> we prepare solution from human serum diluted in wash buffer 1:2, as my
> understanding to why we do this step is to block unwanted proteins in the
> tissue
> so that we prevent background staining, but this step is done before adding
> primary antibody, wont this step block the antigen too since the antigen
> is a
> protein too?
> the other theory i hired is the proteins tend to coagulate together so we
> add
> this solution to dispense them, primary antibody then can easily attach to
> the
> antigen.
> we have two histotext books in the hospital and i red the IHC
> sections, their
> was really nothing clear about why we do this step and i searched the
> Internet
> with no satisfactory answer.
> As u can see i need help!
> step #3 primary Ab
> step#4 link system(secondary Ab against primary Ab (protein)
> Step #5 chromogen DAB
> Am doing IHC for a year and 8 months, till now am confused about this step
> and
> when i train people am not sure what to say to them about it.
> help me please
> Have a great holy day
>
> Wassan
> Histotechnician
> Shorsh hospital
> North of Iraq
>
>
>
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