Dear All,
I wish to perform WISH on an adult zebrafish sample which is treated with collagenase and later acid-fixed (Formaldehyde + Acetic Acid). But before the WISH procedure, I would like to perform Whole-Mount IF as most of the antibodies don't work after the WISH procedure. I generally use FastRed as a substrate for WISH and I perform hybridization and washing at 65 degrees. I am wondering if I can combine both the procedures as they are. I would also like to know if acid fixation causes any problem for the epitope. Once the IF is done, how stable the antigen-1°Ab-2°Ab complex is for further processing.
Thank you.
Hallo Chaitanya, I am not rearly sure what you are meaning when you are talking about WISH. Normally we differnciat betwee FISH (Fluorescence ISH) or CISH (Chromogene ISH --> detection by Peroxidasse DAB or Alkalic phosphatase Fast Red).
If you just need a nucleare stain for better orientation you can use DAPI for fluorescence or haematoxyline for brightfield. If you like to use a special antibody for nuclear staining then you have to try it weather it works better before or after ISH. Formalin fixation per se does not affected the antibody binding because most of commercialy available antibidies are working on formalin fixated paraffin embedde tissue (FFPE). Exceptions are mentioned in the data sheet.
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