[Histonet] counting positive cells in IHC slides

[Favara, Cynthia (NIH/NIAID)]

I do not think this is a silly question but points out the need for a person
trained in the normal microscopic evaluation of tissue to carefully review
the slides, and perhaps suggest alternative stains or tests. In my work this
used to be a pathologist and now I see many people wearing multiple hats and
pathologists have mostly turned into managers along with the rest of the
highly trained and skilled medical personnel.

Probably not a good place to go the beginning of the week!

Just my own thoughts!

c

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317


-----Original Message-----
From: Subratab [mailto:subratab <@t> bdonline.com]
Sent: Friday, August 29, 2003 2:07 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] counting positive cells in IHC slides


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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