[Histonet] counting positive cells in IHC slides

[Subratab]

Dear All,
I am sorry to bother you with a very silly question. I am new in the field
of IHC.
I have stained rat renal tissue slides for macrophage (ED1) with DAKO
EnVision detection system. Now I have to count number of macrophages (ED1
positive cells) per 100 glomeruli. My problem is that the all positive
stained cells are not similar to look; some cells are typical cell-like with
bright red color, but other cells are a bit different in shape or size or
color. So I am in problem in identifying true positive cells. I like to ask
you if there is any systematic way to identify true positive cells from
false staining area or artefact.
ED1 antibody binds with cytoplasmic lysosomal membrane-associated antigen. I
treated my slides with 0.3% triton x100 to break the cell membranes for
better penetration of the antibody. So I think that the shape of the
positive stained cells may be changed and all of them may not look typical
cell-like structure. Am I correct? Can you please explain in some detail
about the change of shape/size/color of the positive stained cells in IHC
slides after staining. Particularly when the antigen is cytoplasmic. Please
let me know if there is any website discussing this issue. Thanks in advance
Dr Subrata Biswas, MD
PhD student, Nephrology Div of Internal Med,
FCM, University of Campinas, SP, Brazil.