[Histonet] Inflammation cell marker on Tissue & Quantitive Immunohistofluorscence

[Pritchard, Michele]

I have a few suggestions that could help.  I am interested in both
inflammation and fibrosis in liver and have looked for markers of these
two pathologies in several different ways.

You say you have used ED-1, this suggests to me that you are likely
staining rat liver.  If you ever use mouse, the absolute best marker for
Kupffer cells, liver resident macrophages, is F4/80.  I have been using
an antibody for F4/80 from Serotec for many years with wonderful
results.  

For assessing neutrophils, I would suggest two different methods, IHC
using a NIMP-R14 Ab (AbCAM) as well as a histochemical stain for
chloracetate esterase.  

To get a bulk assessment of total leukocytes in the liver, you could try
CD45 (CLA, common leukocyte antigen). This will not break down the
various immune subsets, but it certainly can tell you if there is an
increase in total leukocytes (as one would expect during inflammatory
response).

If you would like me to send you a list of markers for specific
lymphocyte subsets, please let me know and I will be happy to furnish
you with some ideas.  However, when most people think inflammation, the
first cells they think of are neutrophils...and then, of course,
macrophages.

ICAM (intracellular adhesion molecule)-1 is upregulated on hepatic
sinusoidal endothelium with inflammation, and it thought to play an
important role in extravasation of neutrophils from hepatic sinusoids
into the parenchyma.  I have used an ICAM-1 Ab from R&D systems to do
this (in mice).

We routinely perform TNFa IHC in liver.  Strikingly, we find that
hepatocytes as well as Kupffer cells can produce TNFa.  I can give you
info on the Ab we use for this immunofluorescence (IF) if you would
like.

As far as quantification goes, our lab quantifies TNFa IF using ImagePro
software and uses intensity of staining. 

It is also possible to quantify individual fluorescent cells with
ImagePro.  Simply speaking, you 'educate' (program) the software to
count for you.  You can achieve this by telling the program what colour
pixels = positive.  This program will also allow you to determine
intensity of each individual spot if you would like;  I don't know if
ED-1 expression changes with activation of macrophages, so I don't know
if intensity measures would help you or not.  With respect to the
quantification, once counted, you can edit what it counted to remove
non-specific staining that happened to fall within the fluorescent
parameters you set.  For example, sometimes, a speck of non-cell
associated fluorophore shows up, I remove these spots.  To help in this
regard, DAPI staining is used; if any given cell contains a nucleus and
my marker of interest, I count it...those random fluorophore bits are
anuclear which gives me rational to untag them.  This is a *MUCH* faster
way to enumerate cells than counting by hand.

I hope this helps, if you have further questions, concerns, want
additional details, please feel free to contact me.  I could discuss
this for days but don't want to bore you if I am not addressing your
specific questions.

Kind regards:

--->mtp


Michele T. Pritchard, Ph.D.
Research Associate
Nagy Laboratory
Department of Pathobiology/NE40
Lerner Research Institute
Cleveland Clinic
9500 Euclid Avenue
Cleveland, OH 44195
 
phone:  216.444.8613
fax:  216.636.1493
 
email:  pritchm <@t> ccf.org
 
Lab location:
Lerner Research Institute
NE4-214

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of TF
Sent: Wednesday, February 25, 2009 12:48 AM
To: histonet
Subject: [Histonet] Inflammation cell marker on Tissue & Quantitive
Immunohistofluorscence

Dear All:

Just wonder any one have the experience to work on inflammation on
different tissues, especially Liver, SKIN, and Brain?
We want to look at wound-resulted inflammation level in these tissues,
using immunohistochemical techniques rather RT-PCR / WB on cytokine
levels.
Can anyone recommend different cell markers for these tisses,
separately?

Another question is how about the Quantitive Immunohistofluorscence. I
have some sections stained with Ed-1, marker of macrophage. It is very
hard to count the number of positive cells ...can we use
immunofluorescence instensity (same exposure time) or the area of
positive region as the quantitive index? At least semi-qunatitive.

2009-02-25 



TF 
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