whole mount in situ hybridization on fish embryo - (Dec/12/2005 )

Hi all,

I'm new to in situ hybridization and would like to hear some of your opinions. I want to detect the gene expression patterns in pigment cells of zebrafish embryos.

1. Which are the important steps that I should take in consideration first when I optimize the ISH protocol? time of fixing? time of proteinase K treatment? probe concentration?

2. Can i store 4% paraformaldehyde at-20C for few weeks? Or must prepare freshly prior fixing? Actually i found some white particles in my paraformaldehyde after 3 weeks storage time at -20C.

3. Should I add DMSO in the paraformaldehyde too?

4. Pigment cells are located on the surface of body. Should I treat the embryo with proteinase K or not?

5. Should I add levamisole in the staining solution for signal detection on pigment cells?

Thanks

To answer your questions, first, always make 4% paraformaldehyde fresh. DMSO is not necessary for the PFA. Protinase K treatment is necessary for the probe to enter the cell. I found 10 min in PK is good. Make sure to fix the embryos for 10-1hr after PK treatment, or they will fall apart. If your background signal is very high, levamisol can help. Some detection kits already have levamisol in them (Fast Red).