Dear Reiko Hanada san, how are you doing today?

Does the antibody you are using is monoclonal or polyclonal? If it is monoclonal, it very specific for mouse. Thus, it will not work in zebrafish. Look for the epitope that the antibody recognizes and compare with the zebrafish sequence. You can do this using the protein blast from NCBI. If there is no match, this antibody does not work in zebrafish. You can do the same thing if you find other antibodies for your experiment.

You can look for a polyclonal antibody or an antibody that has an epitope that is shared between mouse and zebrafish. If there is no commercial product available, you can order in some labs to synthesize antibody for your experiments, unfortunately is expensive to produce a new antibody.

The immunostaining procedures 4 to 5um thickness sections will work fine. There are some methods of antigen retrieval, that depends on the fixation procedure. For Formalin or Paraformaldehyde fixation and paraffin embedded samples, you can use a pH 6,0 Citrate buffer in pressure cooker or microwave. The idea is keeping the sections above 95°C for 10 minutes to break the hydrogen bridges created by fixation. In microwave is more easily to perform, just take care with the hot liquid. In the pressure cooker after reach the temperature just measure the time. After the time, wait the section cool down before going on the protocol. You can adjust this time of the citrate buffer or antigen retrieval if is required.

The blocking steps is also very important to avoid background stain and cross reactivity in both immunohistochemistry and immunofluorescence. Do not forget to perform positive and negative controls with the samples. Well, I hope this could help you. Good luck.

Dear Tulio,

Thanks for the useful information, and I really appreciate you to show us the details about not only antibody but also antigen retrieval, thickness and blocking issue.

We will try for these!!