Thisse Lab - In Situ Hybridization Protocol - 2010 update
Comments
I agree with this comment, the protocol should be fixed by increasing the final volume to 100 ml (to reach the standard AP buffer concentrations).
I use a protocol similar to this and obtained good results before. But rescently, my
colleagues and I are puzzled.
Because in our experiments, everything goes well before the detection of the second probe
(the dig-probe). We still can got good results with the red probe but when we added the BM
purple to visualize the dig-antibody, the purple blue always covered the red areas where it
should not be. It seems not due to the probes because we met the same problem in our
individual opeartions and the probes used are different and worked well before. May it be
the problems of the dig-antibody we use?
If anyone has some answers or better explainations please reply this or contact me(min-
zhang05@mails.tsinghua.edu.cn). It will be very appreciated hearing that.
Zhang, it could be because the AP activity in the first anti-fluorescein probe is not in activiated completely.
1 M glycine pH2.2 works very well in our lab.
Tong
I have been doing in situ hybridisation on zebrafish embryos using a method based on this protocol, however I always obtain staining all throughout the embryos even after only 15 minutes incubation in the staining solution. What could be the cause of this? The whole embryo is purple when using both anti-sense and sense probes.
Thank you for this detailed protocol. One clarification: I believe that the Alkaline Tris buffer recipe has a small error- to achieve the final concentrations listed, you must increase the final volume with H2O to 100 ml, not 50 ml as listed. Alternatively, the salts are supposed to be twice as concentrated as listed.
James Gagnon
jgagnon@fas.harvard.edu