Re: [Histonet] counting positive cells in IHC slides

From:John Kiernan

Your question is not silly. 
You have 2 problems:
  1.  Identification of true macrophages.
  2.  Quantitation of macrophages numbers per renal

I cannot help with Problem 1. Your supervisor should
look at your slides and show you how to recognize
false-positive objects.

Problem 2 is more profound. It assumes that you can
correctly identify a macrophage.  Renal glomeruli are
much bigger than macrophages, and the thickness of the
sections will influence the apparent abundance of
macrophages. In a thick section you will see more
macrophages per glomerulus than in a thin section. You
need to consult with a statistician BEFORE DOING ANY
QUANTITATIVE WORK in this field.

If you don't formally consult your institution's
experts your  work will be rejected by scientific
journals, and nobody will ever know what you saw.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

Subratab wrote:
> 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.
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