I want to identify neutrophil in rat lung tissue by immunohistochemistry.
What is the best marker for identification of neutrophil in lung tissue by immunohistochemistry?
Ly6G is considered to be the most reliable marker to identify neutrophils. However, you have to keep in mind that Ly6G is also expressed in monocytes and granulocytes. That is why the combination with CD68 a and CD15 is expected to be important.
I would like to add some information in terms of the function of neutrophils. Neutrophils respond to inflammation site through three classes of surface protein: selectins, integrins, and chemokines. To resist shear forces within the blood vessel, a neutrophil initially binds the endothelial cells through selectins and undergoes a transient, rolling adhesion to the blood vessel. Integrins expressed by neutrophils change shape in response to activating signals such as chemokines, causing the neutrophil to decelerate from rolling to a stable arrest. Finally, the combination of chemokines and integrin engagement results in a second activation that causes the neutrophil to transform from a spherical circulating cell into an oblong migratory cell.
Furthermore, I would like to emphasize that tumor-entrained neutrophils also express Ly-6G in much the same way as steady state and inflammation. I have once performed tissue immunostaining with Ly-6G antibody (Clone 1A8) to detect neutrophils which have migrated to the breast cancer metastatic foci in the lungs. Tumor-entrained neutrophils (TENs) inhibit metastatic seeding in the lungs by generating H2O2 and tumor secreted CCL2 is a critical mediator of optimal anti-metastatic entrainment of G-CSF-stimulated neutrophils. Most importantly, Ly-6G-positive cells, most of which are neutrophils, express CD11b and MMP9 in the metastatic site.
Ref; Tumor entrained neutrophils inhibit seeding in the premetastatic lung (Cancer Cell. 2011 Sep 13; 20(3): 300–314.)
Staining for either MPO and Ly6G will work provided you have good antibodies.
Rabbit Polyclonal anti-Rat Neutrophil Antibody (FITC) LS-C348005 work well in rat cornea!
Use MPO. Please see the article.
https://science.sciencemag.org/content/sci/suppl/2018/09/26/361.6409.eaao4227.DC1/aao4227_Albrengues_SM.corrected.pdf
I have CD15, CD16, CD45 and CD68. And we are unable to puchase MPO and Ly6G. then which one is more specific use for neutrophil in rats.
Depend on species specific, MPO is good for Human, LY6G is good for mouse. couldn't find good marker for Rats yet
where do you get the LY6G antibody against mouse from for IHC on paraffin sections?
Triet Bui thanks for that advice! But how exactly would you use them only for neutrophils? As far as I read read they mark MDSC cells? Combine with Ly6G? (in mice)
MDSCs are just immature neutrophils if you look into their nuclear morphology. A lot of people think they are just mixture of immature myeloid cells getting out early from BM. Their S100A9 should be lower then mature neutrophils. Also, MDSC has comparable Ly6G (in mouse) as expected from immature neutrophils
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