Hi guys.
I keep getting staining on void area of tissue (breast tumor tissues).
As you can see both in my negative control(without primary antibody) and positive control, there are stretch lines.
I thought that it is due to the quality of my primary antibody but negative control also has same stretch marks even if the staining intensity is low.
It has been also confirmed that it is not attributed to antigen retrieval process since I observed this both in TE buffer treated samples and sodium citrate treated samples
I suspect that something deposit on void area (in between cells) during staining, which enables secondary antibody to accumulate and react with DAB.
Have anybody observed this before? Would using double distilled water instead of water to make wash buffer (TBST) make it better?
Please let me know if anybody observed it before and know how to troubleshoot.
Thanks!
I think there is no responsibility from the antibody, since as you have highlighted, lines are present in the slide stained without primary antibody. There might ay be problems during the preparation of the histologic material such as inclusion or de-paraffination, or even cutting. It would be interesting to check if these lines are also present on the stained glass slides with hematoxylin-eosin
I recommand to take sectoins from another tissue block. Before you start your immuno make a normal HE staining to get informatoins about inclusions other irritating tissue componants. Helpfull could be to stain the sections with PAS to exclude any kind of mycosis.
It could be due to how you section your tissue. If the tissue is not sectioned properly then it could have small tears or holes in it that would accumulate antibody like you are seeing. Do you typically see these small tears in your tissue or is this a recent development? Do others have this issue in your lab?
The strech amrsk are also highlighted as well (I would guess) due to the setting of the substage condensor on your microscope. Either it needs to be raised a little , so that the light is "focused" for the objective, and/or the iris needs adjusting. This would normalise the "contrast" and not make them so obvious. Also if you do just a "H" stain (no IHC reagents and no E) and you still have background, then it could be an endogenous pigment(s).
Thank you so much for all your considerate thoughts on it. I think this happened because of improper sectioning as Matthew mentioned. Now it got resolved. Thanks again!
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