[Histonet] immunohistochemistry fish tissue - help

[Tamara Franz-Odendaal]

I have two questions:

1) Does anyone have a good protocol for immunohistochemistry working with fish tissue embedded in paraffin wax? 
2) Any tricks with working with collagen antibodies (apart from hyaluronidase treatment)?

I have been trying without success to do the above, I have tried an Alexa 488 secondary as well as a horse radish peroxidase secondary. Neither are working and I am wondering if it is something peculiar to fish tissue.  What are good blocks to use to prevent autoimmunofluorescence, or what secondary do you suggest. I am getting high backgrounds due to secondary.  And also no signal in the area of interest (cartilage) so the primaries are also not working well. I am starting to wonder if the antibodies are at fault but before I order more wanted to know if anyone has any good working protocols.

I am working on larval tissue not embryonic. zebrafish.

Or do you suggest whole mount immunohistochemistry?

  Thanks
        Tamara

Dr. Tamara Franz-Odendaal
Dalhousie University
Halifax, Canada