[Histonet] positive controls - adequacy?

[Satoshi Akima]

Dear Listmembers,

this is my first post on the list and I was hoping to canvass some 
opinions.

I am doing some research and would like to stain for neutrophils, 
lymphocytes and macrophages (to see which cell type seems to have a 
pathogenic role). The question is what is good enough as a positive 
control, especially when you are using commercial antibodies for which 
a published body of literature supporting the specificity of the 
antibody is out there. Would you:

A. Just use a blood smear (whole blood)
B. Use white cells from the Buffy coat
C. Use a column (like the miniMACS system using antibody couples 
magnetic beads) to separate out lineages
D. In the case of mononuclear cells use Lymphoprep to separate out PBMCs
E. Culture your PBMCs to get a purer isolate of macrophages

Option C is nice but would cause a big time blow out in the budget - 
all just to get a positive control for IHC. Seems a bit over the top. 
I'm also in a hurry to get my paper out and time management is an issue 
(reagents take time to order in from overseas).

What do people think?
Thanks for your help.

Toshi Akima
PhD Student
Centre for Transplantation and Renal Research
Westmead Millenium Institute
Sydney, Australia