AW: [Histonet] IHC theoretical question

[Gudrun Lang]

The normal blocking serum should be from the species of your secondary
antibody. 
The unspecific binding sites are occupied by the proteins (eg. electrostatic
binding), so the primary antibody can't attach. But the specific
antigen-antibody reaction should not be affected. So this results in primary
antibody on your specific epitops and "secondary" proteins covering the rest
of negativ-charged tissue elements. The secondary antibody binds to the
primary-fc fragment and doesn't mind other proteins of it's own species.

If you use commercial antibody-dilution-solution, the normal-serum-step can
be omitted, because they usually content BSA to block unspecific bindings.

Bye
Gudrun


-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von wassan
alkadhumi
Gesendet: Freitag, 24. Dezember 2010 17:30
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] IHC theoretical question

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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