Dear Colleagues,
My project involves determining position of gene within the nucleus. My preferred technique would be in situ hybridization.
Does anyone have a working protocol for doing in situ hybridization to genes (DNA level) in larva and adult stages?
Also, What would be the best way to prepare the probes for in situ hybridization (kit? company? etc.).
Thank you in advance.
Regards,
Yossi Gruenbaum
Hi Yossi -
For DNA in situ hybridization in any tissue, it’s essential that the probes be fragmented into small (<150 bp or even less) fragments and highly labeled. There is a variety of methods for doing this, but my lab still uses the methods that I’ve published in various places for 3’ end-labeling using terminal deoxynucleotidyl transferase (TdT) (see http://www.springerlink.com/content/u16666g42005364x/fulltext.pdf). Sample preparation is also key, and will depend on which tissue you plan to analyze; formaldehyde fixation (+ permeabilization) is essential to preserve chromosome structure and permit probe entry. You can use the sample preparation methods described for hybridization to gonads in the chapter referenced above as a starting point, but for whole larvae or adults, you will probably need to adapt other sample preparation protocols for IF or RNA FISH to find appropriate permeabilization conditions. Let me know if you have any questions.
Best wishes,
abby