Hello everyone. I am trying to perform in situ hybridization and immunohistochemistry on whole mount larval zebrafish/ dissected larval zebrafish brains (usually ~7dpf). I was wondering if I needed to perform in situ before IHC, or if I could do the other way around?
The reason I ask is because when I perform IHC after in situ for one of my antibodies (dsRed), there is no detection; but performing IHC alone works.
Any help/ protocols/ tips/ advice are appreciated!
Actually, FISH is usually conducted as a more confirmatory procedure over IHC. It makes use of tagged strand of DNA with fluorescen dye, thus makes it a separate technique entirely different from IHC whose principle is hinged upon detection of protein using antibody.
The two techniques are not to be run concurrently, I wondered how you mixed them together. On a better note, I am more conversant with human tissue and would probably suggest you contact your reagent vendor.
Again, don't forget to consult your data sheets attached with your kits, hopefully and usually they recommend tissue types that goes with their kits. Again, this final piece may be useful. Contact previous studies in your research line on the internet.
In situ first and then IHC, but it's possible that your antigen is not preserved through in situ treatment. Check if blocking reagents can interfere with detection masking your epitope, you can eaven try to perform ibridization and washes and then put you antibody before the in situ detection steps, it may works. Good luck !
I agree with Mariarosa. In situ first. Some antigens don;'t work after the ins situ but other are ok. I had good luck with anti GFP detection after the in situ.
Hi, I have done IHC first and then ISH with FastRed. I did this on the oocytes and I acid-fixed the oocytes for this experiments. It works really well for most of the Abs I have used. I am not sure how well the acid fix method would work on 7 hpf larvae. You can give it a try. I used 5% of Formaldehdye (Consider 37% stock as 100%) in 1X PBS and then quickly add 8-10% of glacial acetic acid. Keep your tubes on a shaker for 2 hours and wash in PBS-T. After that you can directly use them for ISH or IHC. When you combine IHC and ISH, make sure to add RNase blocking reagent in your blocking solution when you preform IHC first and later ISH. When you perform ISH after IHC, fix after the 2ndary washes with PFA quickly (10 mins) and add prehyb buffer preheated at 60 degrees. Continue with your normal ISH procedure. Let me tell you that this procedure did not work well for me with TSA system but you can give it a try. Good Luck.
Please refer to the following articles -
Article Fixation/Permeabilization: New Alternative Procedure for Imm...
http://dev.biologists.org/content/early/2018/10/10/dev.167023
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