I'm looking to immunostain some brain tissue collected previously. The animal was *not* perfused prior to tissue collection.
Will the presence of blood interfere with all staining methods (HRP-conjugated, fluorescent, etc)? Or can quenching endogenous peroxidase activity / fluorescence allow for accurate staining?
Sebastian Schmitt
Hi Sebastian,Thanks for getting back to me. Do you know if the lack of perfusion also interferes with HRP staining?
I understand the need for perfusion and fixation, but it simply wasn't done. I am trying to use these tissues in whatever way I can because I cannot change how they were prepared in the past animal studies.
I agree with Sebastian Schmitt
it unfortunately a critical step. It is hard to give a general answer without more details: Are you planning on cutting frozen or embed in paraffin? What is your thickness of cut 7um? (paraffin) or 30-40 um frozen? What are your targets of interest? It may be salvageable if you are using a thin slice of paraffin embedded tissues and looking at clearly identifiable targets like pyramidal cells and membrane bound proteins. Thicker slices and more nebulous targets are going to be harder to differentiate from blood contamination.
Thanks for getting back to me Jeffrey P Cheng The tissue samples were fixed in formalin and is either already embedded or will be embedded in paraffin. I plan on cutting thin slices (5-10um).
For the HRP, I need to stain some pathogens for general localization. In another experiment, I would like to colocalize transmembrane or other cell markers (microglia, astroglia, etc) along with the pathogen with IFC, but I may have to find an alternative strategy.
Selective targets using an HRP / DAB with or without nickel you may be able to perceive general localizations. Colocalization with other cell markers will most likely not be possible due to several factors. With the thin slices you should be able to differentiate your targets vs red blood cell contamination. However, your images may not be very nice looking (darker in appearance). At high magnification you should be able find an area that is not contaminated with red blood cells for images you desire. Any automation or low magnification (counting etc) may be more challenging depending on your software and microscope techniques.
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