We are doing a urinary tract infection project using mouse as an experimental model. We will be doing fluorescent staining (DAPI, DTAF, mCherry). I have several questions:
1) Do I need to infuse the bladder with NBF, or just immerse it? If immersed, will enough formalin get into the bladder to fix the urothelium?
2) If I use Carnoy's solution (which is supposed to preserve mucins), will it interfere with fluorescence?
3) Will formalin harden up enough (if infused and the bladder urethra tied off to prevent leakage) to view the bladder in a distended condition?
Hello,
Hope I can help you with your questions.
1) Depends on how long you incubate in formalin the mouse bladder is rather small so eventually the formalin will reach the inner layers and urothelium and fix it. However I think you will have the best results if you perfuse the whole animal with formalin.
2) Depends on your fluorescent dyes. I think Carnoys is acidic and different dyes are more or less sensitive to pH.
3) I do not think formalin will harden (though the tissue will, a little bit), maybe you mean paraffin? We used to infuse the bladder with cryosectioning media before freezing them for sectioning when we wanted to have sections with distended bladders and it worked fine. I think you could try to do the same thing with paraffin.
Have a look at the methods section in our papers it might be helpful :-)
Best regards
Karl Svennersten
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