Hi Everyone,
I was hoping that someone had tested out the Stellaris smFISH protocol. How does it compare to the Ji & Oudenaarden, 2012 protocol on wormbook? Specifically has anyone used Stellaris hybridization and wash buffers?
Thanks!
I had good luck with the Stellaris smFISH protocol when probing for GFP transcripts in dissected germlines. I followed their protocol (the “C. elegans larvae version”) with only small changes to accomodate the volumes I was working with and purchased their buffers and pre-labelled oligos to get everthing working with minimal fuss.
In addition to the protocol from Ji & Oudenarden, I found the detailed protocol from Christian Lanctôt’s lab very useful: https://www.ncbi.nlm.nih.gov/pubmed/26564238
Thanks for the tips!
I figured I would post an update in case anyone was curious. I used the buffers from biosearchtech and they did the trick. I found that it was better to use acetone for a fixative rather that formaldehyde (however, I was not using GFP or similar fluorophores) to reduce background signal as stated in Bolková & Lanctôt, 2015. Additionally, the inverted chambered coverslip was a pain and did not work on our inverted microscope, so we just switched to using 1.5 mL tubes (we were assaying L1s so the baskets would not work). We also increased [probe] to 250 nM and used Prolong Gold (also in Bolková & Lanctôt, 2015) instead of the Vectashield mounting medium recommended in the biosearch protocol.