Double staining protocols for Insitu hybridization and immunohistochem - (Nov/15/2000 )

Dear sirs,I am currently working with in situ hybridization (ISH) protocols using DIG-labelled cRNA probes on paraffin-embedded tissue blocks.I would like to identify the cells in my tissue biopsies which express the cRNA probes. I would appreciate it if you could send to me any existing protocols on simultaneous detection of cRNA probes by ISH and cellular antigens by immunohistochemistry.Thanking you in advance for your help,Georgina Xanthou.

There are many ways to do multiple rounds of staining with different substrates. The easiest is just to do multiple rounds. Simply stain with one antibody/substrate combination, then restain with a second antibody/substrate, using a different substrate of course. The only thing to remember is that if both antibodies are linked to the same enzyme, e.g. alkaline phosphatase, or if both primary antibodies were generated in the same species, e.g. mouse, some activity will remain from the first round and will also stain up in the second round.There are two ways around this. The most simple is to develop the first round with the DARKER colored substrate (e.g. blue or purple) and the second round with a light colour (e.g. red AP or orange HRP). This means that even if your blue cells stain a little with the red substrate you will not be able to see it.