[Histonet] Background staining: mouse on mouse Broad spectrum kit

From:Cathy Malcontenti-Wilson

Dear All,
I really need some advice regarding an ongoing intense background problem.
We use FFP sections of mouse liver.
We are using an immuno kit called Zymed Histomouse Max Kit (It is a HRP- 
DAB one).
Our primary ab is mouse anti human VEGF (1:1000).
The kit is called a BROAD SPECTRUM kit because it is meant to be able to be 
used for mouse, rabbit, rat and guinea pig primaries.
The secondary antibody is not completely explained in the kit but I think 
it is of goat host species.
Question: If it is broad spectrum does this mean that the secondary 
antibody is a mixture of different antibodies?
The kit contains its own non specific block, with no details of what it 
actually is.
Question: If the secondary ab is raised in goat, is this non specific block 
normal goat serum? If not, what could it be?
We know that when I incubate the tissue with DAB only, we get endogenous 
peroxidase staining which is pretty much abolished with H2O2 block.
We know that when we omit the primary antibody and keep all the other 
layers, we get intense background staining of the sinusoidal (endothelial) 
lining of practically all the sinusoids, and we get brown staining in 
plasma cells, macrophages, connective tissue and other cells within vessels.
Question: Omission of non specific blocking reagent showed increased 
background staining. Does this mean that the blocking agent is actually 
blocking some of the background but not all? Why not?
We know that when we omit both the primary antibody and the non specific 
block then we get background staining.
Question: What does this mean?
We know if we omit the secondary antibody only, we get no staining.
Question: What does this mean?
I would greatly appreciate any input into this problem which has been 
plaguing us for a while now.
I hope someone can help, I cant beleive what a great wealth of knowledge 
there is on this histonet list.
Cathy Malcontenti-Wilson

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