[Histonet] counting positive cells in IHC slides

[Philip Oshel]

The Gundersen refs are excellent and widely used. I would also like 
to add a basic text in stereology: John Russ' "Practical Stereology". 
Note that this is for the 1st edition. The 2nd edition, recently out, 
has a co-author whose name I forget, sorry.

Phil

>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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-- 
Philip Oshel
Supervisor, BBPIC microscopy facility
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison,  WI  53706 - 1284
voice: (608) 263-4162
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