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: prit...@ccf.org Lab location: Lerner Research Institute NE4-214 -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet =================================== P Please consider the environment before printing this e-mail Cleveland Clinic is ranked one of the top hospitals in America by U.S. News & World Report (2008). Visit us online at http://www.clevelandclinic.org for a complete listing of our services, staff and locations. 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