Re: [Histonet] counting positive cells in IHC slides

From:Cathy Gorrie

Dear Subrata,

I agree entirely with J.Kiernan's reply.

However there are a few steps you may want to take to assist in 
identifying what your antibody is staining.

You don't mention whether if not you used control sections as well. I 
would suggest a series of at least three.

1. Test tissue with primary omitted. This will show any staining by 
the cross reactivity by the secondary antibody, any background 
staining and/or auto fluorescence. Ideally this control should appear 

2. Test tissue with secondary omitted. This will show any 
autofluorescence in the tissue. Ideally this control should appear 

3. Tissue known to be positive for macrophages, previously identified 
by other means. This will confirm specificity of your antibody (ED1). 
Ideally this control should appear POSITIVE for macrophages only

These controls will help you establish the range of normal staining 
patterns for you macrophages and for your tissue. Treat your control 
sections exactly the same as your test sections except for the 
omission of antibody.
The only thing staining positive should be your macrophages. If there 
is something else coming up positive you need to be able to identify 
and/or distinguish between it and macrophages.

Cheers, Cathy

Catherine Gorrie
School of Medical Sciences
University of New South Wales
Sydney, N.S.W. 2052

Phone: 61-2-9385 2462
Fax  : 61-2-9385 8016

30/8/03, 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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