I wholeheartedly agree with John Kiernan's advice on statistical
considerations.  You have to remember that in any counting scheme like the
one you mention (counting the number of macrophages per 100 glomeruli)
you're estimating cell number and you must have a method for achieving an
unbiased estimation.  The best way to achieve unbiased estimates is by
employing the stereological methods outlined by Gundersen et al. Before you
begin, you should consult the following papers:

Gundersen HJ, et al. The new stereological tools: disector, fractionator,
nucleator and point sampled intercepts and their use in pathological
research and diagnosis.  APMIS. 1988 Oct;96(10):857-81. Review. 
 
Gundersen HJ, et al. Some new, simple and efficient stereological methods
and their use in pathological research and diagnosis.  APMIS. 1988
May;96(5):379-94. Review.

These papers are the seminal references for the most commonly practiced
stereological methods.  They should help in the design of your counting
procedures and aid in the statistical analysis of the data.  They are a must
read for anyone attempting to estimate pretty much anything from microscopic
sections.

Scott Turner
DNAX Research Institute
Palo Alto, CA


> 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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