I am trying to label extracted drosophila larvae brains by fluorescent in situ hybridization with fast red. Our lab has previously done plenty of in situ hybridizations on zebrafish embryos with success, but I'm afraid the fly larvae brain is a whole different beast. Our attempts using both NBT/BCIP and Fast Red with two different probes so far have given little if any discernible signal within the brain that may just be background, but really dark labeling in tubular structures near the surface that I believe are the vasculature with both the antisense and control sense probes. My first thought was I needed to increase the Proteinase K concentration to increase the probe penetration into the tissue, so I followed a protocol with a stronger ProK concentration and longer incubation time (being careful not to destroy the tissue of the brain), but I really didn't see any difference. So two main problems: dark non-specific vasculature staining is interfering with my ability to see and detect within the brain, and my probes don't seem to be giving much if any signal. I feel like if the vasculature regularly non-specifically stains, there must be a trick out there to minimize that staining. Any help out there? Thanks!
Presumably you will see the respiratory tubes. Drosophila goes to different stages of larvae, each different. Try microwave techniques: se Mathilde Boon's book on microwaving or look into Method special edition on microwaving, you will find it by E.Marani as special editor. Moreover insect nervous tissue is frequently a problem.
Hope this helps you out.
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