Dear all,
I try to identify the cell type (lamina propria) bearing a receptor which I stained (DAB, and AlexaFluor-546) on paraffin-embedded human colonic tissue. Previous co-localizations of other antigens in epithelial cells (crypts) worked well (AF-546 and AF-488). I adjusted both antibody titers to a minimum, and still, although I tried more than 20 different markers for lamina propria cells, got no co-localization of the two antibodies' signals.
Is it possible that one staining prevents (e.g., quenches) the detection of other signal? Are there any other decisive issues?
Thanks a lot.
The lamina propria is the part of the mucosa in between the colonic crypts above the muscularis mucosa. There are several different cell types in this compartment including inflammatory cells, capillaries, nerve endings, and fibroblasts/cytes. You need to specify on which of these very different cell types you expect to see your receptor. Then you can read the literature to identify a common antigen on that cell type to co-localize. If you used a co-localizing antigen in epithelial cells/crypts, it is most likely absent in the mesenchymal cells of the lamina propria.
Thank you all for the answers.
Nils, the colocalization study in the epithelium/ crypts was for a different antigen/study, I just wanted to say that this one worked with my staining protocol. Lamina propria cells are stained for a different antigen, and the cell type is unknown. I only co-localization I obtained was with vimentin, hence it should be mesenchymal cells. However, all other markers (cd3, cd4, cd11a, cd11b, cd11c, pgp9.5, a-smoothmuscleactin, cd34,...) did not co-localize. Therefore, I was wondering whether the signal of my antigen of interest is able to quench/prevent staining with another antibody.
SMA and CD34 are for capillaries and endothelial cells. SMA also stains smooth muscle, but thats not present in LP. CD3 and CD4 is for T-lymphocytes. Pgp9.5 is neural. CD11b is an integrin on macrophages. That leaves only fibroblasts. differentiating fibroblasts/cytes stain with vimentin, but are negative for almost everything else. You can try Fibroblast Surface Angigen (SFA), Fibroblast Specific protein 1 (FSP1), procollagen type 1, or prolyl-4-hydroxylase beta. All these are not used in clinical pathology, and therefore are not as robust as lets say CD34 of endothelial precursors.
To complete, I would also do CD45, which covers all lymphocytes and CD138 which covers plasma cells. Also, I would look at the morphology of the cell staining with your antibody. I personally can say with certainty if the cells are inflammatory or mesenchymal fibroblastic, or vascular. It helps to compare serial sections of the immuno with an H&E stained slide.
Quenching of fluorescense is usually not a problem, unless the two antibodies are really close together. But you can use horse radish peroxydase on one secondary, adn alkaline peroxydase on the other secondary antibody and do immunohistochemistry instead of fluorescense. On sections of the colon, this is much better anyway.
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