FAQ

Can the Stellaris® RNA FISH method be combined with immunofluorescence?

Stellaris RNA FISH can be combined with immunofluorescence (IF), although some antibodies may not be compatible with FISH conditions. The fixation method used will affect the integrity of cellular structures and macromolecular assemblies of RNA and protein. It may also affect the accessibility of the epitope to be interrogated. Three approaches have been successful in combining Stellaris RNA FISH with IF: 1) Just adding in the IF-antibody/antibodies into the hybridization and/or wash buffers. 2) Performing the IF, then re-fixing the sample with formaldehyde, and then performing the RNA FISH. 3) Performing RNA FISH, then re-fixing the sample, and then performing the IF.

The following methods article and supplement outlines a protocol to combine the Stellaris RNA FISH method with immunofluorescence in cells, with details on page 4 of the Supplemental Material:

Imaging Individual mRNA Molecules Using Multiple Singly Labeled Probes. (2008) Raj, A.; van den Bogaard, P.; Rifkin, S.A.; van Oudenaarden, A.; Tyagi, S. Nature Methods 5(10), 877-9.

Another excellent paper combining IF with Stellaris RNA FISH is:

Visualization of Single mRNAs Reveals Temporal Association of Proteins with microRNA Regulated mRNA. (2011) Shih, J.D.; Waks, Z.; Kedersha, N.; Silver, P.A. Nucleic Acids Research 39(17), 7740-9.